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1The Renal Section, 2Molecular Physiology and Biophysics, 3Cardiovascular Sciences Section, Baylor College of Medicine, 4Pathology and 5Cardiology Departments, University of Texas Health Sciences Center, Houston, Texas 77030; and 6The Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C, Denmark
Submitted 10 December 2002 ; accepted in final form 19 March 2003
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ABSTRACT |
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calcium homeostasis; phenogenesis contribution; theraputic benifits
Through the evolutionary process from fish to mammals, STC1 appears to have maintained its functional role in calcium regulation, because mammalian STC1 appears to be involved in calcium homeostasis in the normal physiology of the gut (16) and in the adaptive response of brain cells to ischemic injury (28). Because cardiomyocyte calcium homeostasis demonstrates a wide range of abnormalities in patients with heart failure (2, 9, 11, 13, 17, 21), we hypothesized that myocardial expression of STC1 may be relevant to calcium homeostasis in the failing heart.
Our current data suggest differential expression of STC1 protein in cardiomyocytes and blood vessel walls of failing hearts and are consistent with a potential role for STC1 in cardiomyocyte calcium homeostasis.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Materials. All chemicals were purchased from Sigma Chemical (St. Louis, MO) except where stated. Fluorescent probes were purchased from Molecular Probes (Eugene, OR). STC1-specific rabbit polyclonal antibodies (that do not cross react with STC2) and recombinant hSTC1 protein were kindly provided by Dr. Henrik Olsen, Human Genome Sciences (Rockville, MD). Recombinant human STC1 protein was expressed in a baculovirus expression system and is >90% pure (6, 27).
Immunohistochemistry staining. Human left ventricular tissue samples were fixed in 2% paraformaldehyde followed by dehydration in graded alcohols and embedded in paraffin using standard techniques. Five-micrometer sections were cut, dried, and rehydrated for labeling with affinity-purified anti-hSTC1 antibodies, by using an avidin-biotin kit (Vector Laboratories) and peroxidase enzyme-based detection system, with diaminobenzidine as the substrate. Control staining was carried out with normal rabbit IgG and showed no labeling. Photomicrographs were taken using a Zeiss Axioscope microscope (Hertfordshire, UK) fitted with a Leaf Microlumina (ElectroImage; Great Neck, NY) camera system. Staining density was analyzed using ImagePro Plus software.
Rat heart tissue was perfusion-fixed with 3% paraformaldehyde in 0.1 M sodium cacodylate. Before paraffin embedding, tissue blocks were dehydrated in graded ethanol (70% for 2 h, 96% for 2 h, and 99% for 2 h) followed by overnight incubation in xylene. Paraffin sections (2 µm) were cut on a Leica RM 2126 microtome and dried overnight at 37°C. Sections were incubated with rabbit anti-hSTC1 immune serum, and labeling was visualized using peroxidase-conjugated secondary antibody. Control staining was carried out with omission of the primary antibody and showed no labeling. Image capture was carried out by using a Leica DMRE microscope.
In situ hybridization. Synthesis of digoxigenin-labeled (Boehringer Mannheim; Indianapolis, IN) sense and antisense canine STC1-specific probes (22) was carried out using MEGAscript kit (Ambion; Austin, TX). These probes are unique and share no homology with other known calcium channels, including transient receptor potential channels. Paraformaldehyde (4%)-fixed and paraffin-imbedded normal human heart sections were rehydrated and washed for 10 min in PBS. Sections were then treated with 50 µg/ml proteinase K (Promega; Madison, WI) for 12 min in Tris-EDTA buffer (20 mM Tris·HCl, pH 8, 20 mM EDTA) and washed again in PBS for 10 min. Repeated fixation in 4% paraformaldehyde was carried out, followed by treatment with acetic anhydrate [125 µl acetic anhydride added to 50 ml of 0.1 M triethanolamine-HCl (Fisher Scientific; Pittsburgh, PA), pH 8], for 10 min, and a 5-min wash in PBS. Sections were prehybridized for 4 h at 42°C in a prehybridization solution [50% formamide (Fluka; Milwaukee, WI), 5x SSC (0.75 M NaCl and 0.075 M trisodium citrate, pH 7), 1 mg/ml transfer RNA (Boehringer Mannheim; Indianapolis, IN), 100 µg/ml heparin, 1 x Denhardt's solution, 1% Tween-20 (Fisher), 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Fisher), and 5 mM EDTA]. Hybridization was carried out overnight at 42°C in prehybridization solution containing 1 µg/ml antisense or sense STC1 probe. Slides were washed sequentially in 0.2x and 0.1x SSC (carried out at 42°C for 15 and 30 min, respectively), followed by a 20-min wash in PBT [PBS containing 0.2% BSA and 0.1% Triton X-100] at room temperature. Slides were then incubated for 1 h in PBT containing 20% heat-inactivated sheep serum, followed by 2-h incubation with preadsorbed anti-digoxigenin antibody. The slides were then washed for 10 min in alkaline phosphatase buffer [50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20, and 100 mM Tris, pH 9.5], followed by a 10-min wash in alkaline phosphatase buffer containing 0.5 mM levamisole. Signal was detected after a 48-h incubation in alkaline phosphatase buffer containing 0.5 mM levamisole, using nitroblue tetrazolium chloride and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid as substrates. Staining was stopped by a final wash in PBS.
Real time PCR. Left ventricular tissue (apex) was obtained from
normal hearts (n = 6) and from patients with dilated cardiomyopathy
(n = 6). STC1 mRNA level in the myocardium was measured using
real-time quantitative RT-PCR as previously described
(7). Results were normalized to
the expression of the housekeeping gene, 
-actin, in the same sample.
-Actin primers used were the following: forward-primer
5'-CCCTGGCACCCAGCAC-3 and reverse-primer
5'-GCCGATCCACACGGAGTAC-3', corresponding to bases 956–971
and 1008–1128 of human actin mRNA, respectively (GenBank Accession no.
BC014861
[GenBank]
). STC1 primers used were the following: forward-primer
5'-CAGCTGCCCAATCACTTCTC-3' and reverse-primer
5'-TCTCCATCAGGCTGTCTCTGA-3', corresponding to bases 736–755
and 821–841 of human STC1 mRNA, respectively (GenBank Accession no.
XM-011704). Stanniocalcin primers were unique to mammalian STC1 and shared no
homology with STC2.
Preparation of cultured rat cardiomyocytes. Adult rat cardiomyocytes were harvested as previously described (14, 19). Briefly, after anesthesia, rats (average weight of 200 g) were given 3,000 IU of heparin by intravenous injection. The heart was exposed by a longitudinal thoracotomy incision, and the thymus and fascia were cleared from the aorta with a sterile swab. The aorta was cross-clamped and cut distally, and the heart was removed and placed in 50 ml of Joklik's media [1 package of Joklik's media powder (GIBCO-BRL), suspended in 50 ml water, and supplemented with 3.91 g taurine, 2.0 g NaHCO3, 0.391 g L-glutamine, and 0.282 g adenosine]. The heart was rinsed and transferred to fresh ice-cold Joklik's media. The aorta was cannulated and flushed with cold Joklik's media by using a syringe, followed by perfusion with a perfusion pump at a rate of 12–15 ml/min, for 5 min. The heart was transferred to a Langendorff apparatus, flushed with warm Joklik's media, and further digested by perfusion with Joklik's media containing 0.1% collagenase and 0.1% trypsin for 45 min.
The ventricles were minced and placed in digestion buffer containing 0.1% collagenase (in Joklik's media). Minced heart tissue was incubated in a shaking water bath at 37°C for 30 min. The supernatant was transferred to a conical tube and centrifuged (for 3 min at 50 g). The resultant pellet was washed twice in 4% BSA solution and once in 2% BSA solution. The pellet was then suspended in 20 ml Joklik's media (pH 7.2), containing 2% BSA, followed by slow addition of CaCl2 to yield a final concentration of 1.25 mM. The cells were finally pelleted (as above) and suspended by gentle pipetting in 4 ml of warm, serum-free DMEM medium.
The cell suspension (1–2 drops) was then layered onto sterile laminin-coated coverslips. After 30 min of incubation at 37°C in 5% CO2-95% O2 (to allow cell attachment), plating media [DMEM containing 10% fetal bovine serum, 3 µg/ml Ara-C (to inhibit fibroblast growth), 10 µg/ml insulin, and 5 U/ml each of penicillin and streptomycin] were gently added. Cells were fed with fresh plating media every other day (19). The cells began beating after 5–7 days of incubation, and the experiments were carried out between days 7 and 14. Animal experiments and care were in compliance with National Institutes of Health and institutional guidelines.
Measurement of intracellular Ca2+ by fluorescence spectrophotometry. For calcium measurements, we have defined a calcium transient as a spike, or spikes, that increase in intensity to such an extent that the majority of the volume of a cardiomyocyte fluoresces at maximum intensity and is followed by cell contraction.
Measurements of fluorescence intensity of calcium fluoroprobe (Fluo3 at 3 µmol/l concentration) and sequential image recording of contractile events were made on a Wallac/Perkin-Elmer (Gaithersburg, MD) Concord system incorporating a SpectraMaster multiwavelength controller and temperature-controlled stage (Melville, NY). To detail at least one calcium transient, sequential images captured over a 2- to 5-s span were selected, and video recordings of these events were made for a number of minutes (average of 25,000 image acquisitions) using an Olympix AstroCam CCD4100 Fast Scan [12 bit; 768 x 576: 1,000 frames/s; 9-µm resolution; (3)]. Cell contraction studies were performed by using a Wallac/Perkin-Elmer Concord system, employing Fluo3 (3 µmol/l final concentration) as a fluorescent molecular probe. Contractile-fluorescence data were captured with a Merlin High Performance Ratio Fluorescence Workstation, utilizing a SpectraMaster monochromator and Rainbow Multi-wavelength Filter Wheel (Olympus America; Melville, NY).
Whole cell patch-clamp studies. Calcium currents
(ICa) were recorded from single cultured rat
cardiomyocytes at room temperature (20–23°C), using an EPC-9
amplifier [HEKA, Lambrecht, Germany
(10)]. The recording chamber
was perfused with Tyrode solution [in mM: 140 NaCl, 5.4 KCl, 1.8
CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, pH 7.4] at a
rate of 1.5–2.0 ml/min. The pipettes had resistances of
3–5M
when filled with pipette solution [in mM: 130 cesium
gluconate, 10 tetraethylammonium chloride, 10 HEPES, 10 EGTA, 3 MgATP, and 0.3
Na3GTP]. The membrane capacitance and series resistance were
electronically compensated to minimize the capacitive transient and to improve
the dynamic response. Membrane currents were filtered at 3 kHz, recorded, and
analyzed with PULSE & PULSEFIT software (HEKA).
Statistical analysis. The means ± SE were calculated using paired and unpaired t-test as appropriate. Results were considered statistically significant if P was <0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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(25), atrial
natriuetic peptide (ANP), and brain natriuretic peptide (BNP)
(1). The localization of STC1
to blood vessel walls and cardiomyocytes suggests a role for STC1 in vascular
tone and cardiomyocyte function.
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Expression of STC1 mRNA in myocardial tissue, including cardiomyocytes, arterial walls, and fibroblasts. Stanniocalcin expression is ubiquitous in mammals, and its mRNA has been detected in a large number of organs, including the heart (4, 5, 22). Using in situ hybridization, we determined the cellular elements that express STC1 in normal human heart tissue. STC1 mRNA is expressed in cardiomyocytes, smooth muscle cells of the arterial walls, and connective tissue fibroblasts (Fig. 3). To investigate the mechanism of cardiomyocyte STC1 protein upregulation in heart failure, we performed real-time quantitative RT-PCR on RNA obtained from left ventricular tissue samples of patients suffering from heart failure. Results were compared with those observed in the heart tissue of accident victims and showed no difference in STC1 mRNA levels (Fig. 3). Thus our data suggest that upregulation of STC1 protein in heart failure occurs at a posttranscriptional level.
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STC1 slows endogenous beating rate of and diminishes intracellular Ca2+ transients in cultured cardiomyocytes in a manner that involves L-channel inhibition. Because of the technical difficulties and ethical issues that are associated with establishing cultured human cardiomyocytes, we performed the following experiments in cultured dedifferentiated adult rat cardiomyocytes, which have been used as a model for the study of heart failure (19). In the following set of experiments, we measured cytoplasmic calcium transients in cultured contractile rat cardiomyocytes, using fluorescence microscopy (Fluo3). Previous studies to determine the baseline spontaneous beating rate in these cells revealed no significant rate variations, with continuous tracings extending for up to 1 h (19). Initial dose-response experiments showed that STC1 at a concentration of 50 ng/ml or greater provides an equivalent decrease in the rate of contraction through the first 10 min. Thus the remaining experiments were carried out using 50 ng/ml STC1. Treatment with STC1 for 10 min produces a statistically significant reduction (75%) in the frequency of cardiomyocytes contraction compared with heat-denatured STC1-treated cells (Fig. 4A), which was associated with 50% attenuation in the peak calcium rise (amplitude of whole cell Fluo3 fluorescence, recorded with each transient; Fig. 4B). Some of the cells, however, showed partial rate recovery after 10 min but virtually all cells ceased to contract after 25 min, and fluorescence intensity (corresponding to intracellular Ca2+ concentration) approached the background.
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To further define the mechanism of attenuation of calcium transients by STC1, we measured transmembrane calcium currents using the whole cell patch-clamp method. Addition of nimodipine to the bath solution blocks L-channel-mediated calcium currents in depolarized cardiomyocytes. Similarly, addition of STC1 for 5 min provides >50% reduction in transmembrane calcium currents through the L-channels (Fig. 5). This inhibitory effect was significantly diminished after a washout period, suggesting that the inhibitory effect of STC1 on the channel is reversible. In conclusion, STC1 is a potent inhibitor of the L-type calcium channels in cardiomyocytes.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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STC1 is a calcium-regulating hormone in bony fish (8). It maintains calcium homeostasis by inhibiting calcium influx from the aquatic environment through the gill and intestine to the blood stream (15, 24). Similarly, the mammalian STC1 is involved in calcium homeostasis in the normal physiology of the gut (16) and in the adaptive response of brain cells to ischemic injury (28). Thus, through the evolutionary process from fish to mammals, STC1 appears to have maintained its functional role in calcium homeostasis.
Because STC1 is expressed in the heart where calcium flux across cellular
membranes is essential for cardiac function, we reasoned that STC1 might have
a role in calcium homeostasis in this organ, either in normal physiology, or
in the adaptive/maladaptive processes that lead to the heart failure
phenotype. Our current data reveal major alterations in the level of STC1
protein in the failing heart, where it is markedly elevated in cardiomyocytes
and arterial walls. In contrast, STC1 levels are downregulated in these sites
following ventricular unloading with LVAD. Because myocardial levels of STC1
mRNA are not altered in patients with cardiomyopathy, we reason that the
increase in STC1 protein in the failing heart is regulated at a
posttranscriptional level. Furthermore, our data suggest that STC1 may be
added to the list of genes that respond to left ventricular workload and may
resemble TNF-, atrial natriuretic peptide, and brain naturiuretic
peptide in the manner in which it is regulated
(1,
25).
The significance of STC1 upregulation in heart failure is not clear at present. However, our data provide some clues to its function. First, treatment of cultured contractile cardiomyocytes with recombinant STC1 reduces the rate of cell contraction and diminishes the rise in cytoplasmic calcium with each contraction. This effect appears to be mediated at least in part through inhibition of L-channels. Using whole cell patch-clamp studies, we demonstrate that addition of STC1 to the bath reduces transmembrane calcium currents in cardiomyocytes in a manner that is similar to treatment with nimodipine (an L-channel blocker). Inhibition of transmembrane calcium currents by STC1, however, was not complete at the concentrations employed and appeared to lag in time (see Fig. 4), suggesting the involvement of an intracellular signaling cascade in its effect. This possibility is supported by recent findings by McCudden et al. (18) that suggest the existence of STC1 receptors in mammalian liver and kidney. However, we cannot presently rule out the likelihood of a direct effect on the channel or of a combination of direct and indirect (signaling-mediated) regulatory inputs. Through its effects on L-channels, STC1 is expected to reduce myocardial rate and contractility. Thus we propose that upregulation of STC1 in the failing heart may be cardioprotective initially, because it would reduce ventricular workload. However, sustained upregulation of STC1 in the failing heart may become maladaptive, because it could potentially reduce ejection fraction.
Finally, the expression of STC1 in myocardial tissue is not confined to one cell type. In addition to its expression in cardiomyocytes, STC1 is also present in interstitial fibroblasts, smooth muscle cells of the arterial wall, and endothelium; cells that use calcium for regulation of intracellular processes. Together, our findings indicate that STC1 may have a unique role in the adaptive/maladaptive phenotype of heart failure and may contribute to its pathogenesis. Additional studies are needed to further define this role and the suitability of STC1 as a therapeutic target in heart failure.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-42550 and funds from the Renal Section and Debakey Heart Center, Baylor College of Medicine and the Methodist Hospital, Houston, Texas.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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