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Department of Medicine, Division of Endocrinology, North Shore University Hospital; and Departments of Medicine and Cell Biology, New York University School of Medicine, Manhasset, New York 11030
Address all correspondence and requests for reprints to: Dr. Kaie Ojamaa, Division of Endocrinology, North Shore University Hospital, 300 Community Drive, Manhasset, New York 11030.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and
ß-myosin heavy chains, ß-adrenergic receptors, sarcoplasmic
reticulum (SR) calcium-activated adenosine triphosphatase (SERCA2), and
phospholamban (PLB) are regulated by thyroid hormone (3). Cardiac
muscle contraction and relaxation are regulated by the intracellular
free calcium concentration
([Ca2+]i), which is
largely determined by SR Ca2+ release and
reuptake by SERCA2 (3, 4). PLB is a phosphoprotein that regulates
SERCA2 activity (5, 6). The ratio of SERCA2/PLB protein is an important
determinant of calcium cycling kinetics in the cardiac myocyte (5).
Phosphorylation of PLB by cAMP- dependent protein kinase (PKA)
relieves its inhibition of SERCA2 and is considered to be the primary
mechanism by which ß-adrenergic agonists exert positive inotropic
actions on the heart (5, 7). The role of PLB in regulating cardiac
contractile function has been best demonstrated in null-PLB transgenic
animal models that show enhanced contractility that is independent of
both ß-adrenergic stimulation and thyroid hormone status (8). The present studies examined the effects of T3 on PLB expression and its covalent modification by phosphorylation in the hypothyroid rat heart (9, 10). As thyroid hormone is known to regulate myocyte contractility (2) in part by changes in intracellular calcium dynamics (6, 8), these experiments tested the hypothesis that this effect is mediated by changes in PLB phosphorylation and the ratio of SERCA2 to PLB protein in the myocardium (11). The current studies showed that T3 treatment increased both the SERCA2/PLB ratio and PLB phosphorylation concomitant with a significant enhancement of cardiac ejection fraction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An additional subgroup of hypothyroid animals received a single ip injection of T3 (20 µg) 1 and 2 h before removal of the heart for protein analysis.
Tissue preparation
Frozen LV tissue was pulverized using a mortar and pestle placed
in liquid nitrogen, and then homogenized with a Potter-Elvehjen
homogenizer in 10 vol buffer containing 30 mM Tris-HCl (pH
7.6); 2 mM EDTA; the protease inhibitors leupeptin,
aprotinin, and antipain at 10 µg/ml; 1 mM
phenylmethylsulfonylfluoride; 2.5 mM benzamidine; and
Ser/Thr phosphatase inhibitor cocktail (Sigma). Fresh LV
tissue was homogenized similarly. A portion of the homogenate was
centrifuged at 10,000 x g for 30 min at 4 C, and the
supernatants (S10) were retained. Another portion of the homogenate was
extracted with 0.5% Triton X-100 on ice for 30 min. Protein
concentrations of both the extracted homogenate and the S10 fraction
were determined by the method of Lowry. Laemmli buffer was added to
known amounts of protein, and the samples were solubilized at 30 C for
30 min before SDS-PAGE. For RNA analysis, frozen LV samples were
extracted as previously described (12).
Gel electrophoresis and immunoblot analysis
Tissue homogenates containing either 15 µg protein (for PLB)
or 38 µg protein (for SERCA2) were resolved by electrophoresis on
15% or 10% polyacrylamide gels (10 x 7 cm, SDS-PAGE) to
determine total PLB or SERCA2 protein content, respectively. The
proteins were electrophoretically transferred onto nitrocellulose
paper, and nonspecific binding on the immunoblot was blocked using 5%
milk in Tris-buffered saline. Separate blots were incubated overnight
at 4 C either with monoclonal anti-PLB antibody (A1) at 0.25 mg/ml
(Affinity BioReagents, Inc., Golden, CO), which recognizes
both phosphorylated and nonphosphorylated PLB (13), or with monoclonal
anti-SERCA2 antibody (1:500 dilution; Affinity BioReagents, Inc.). After extensive washing of unbound primary antibody with
Tris-buffered saline/milk, the blots were incubated with horseradish
peroxidase-conjugated goat antimouse IgG at a 1:4000 dilution
(Bio-Rad Laboratories, Inc., Hercules, CA). PLB and SERCA2
protein bands were visualized on x-ray film using chemiluminescent
reagents (NEN Life Science Products, Boston, MA).
Resolution of all phosphorylated and nonphosphorylated PLB pentamers in the S10 fraction (15 µg) was achieved by 15% SDS-PAGE (16 x 20 cm) at 25 mA/gel for 7 h (13). Proteins were electrophoretically transferred at 80 mA onto nitrocellulose paper (12 h) and immunoblotted with anti-PLB antibody (A1) as described above. To facilitate the comparison between the phosphothreonine and phosphoserine sites of PLB, 300 µg (for hypothyroid) and 600 µg (for T3-treated) S10 fractions were loaded into 2.5-cm wells and resolved by 15% SDS-PAGE. After electrophoretic protein transfer, each lane was cut into three strips for immunoblot analysis with one of three antibodies: monoclonal anti-PLB (A1), polyclonal antiphosphoserine 16 (PS-16), and antiphosphothreonine 17 (PT-17) PLB antibodies at a 1:1000 dilution (Fluorescience Ltd., Leeds, UK) (14).
For protein quantitation, the immunoblot chemiluminescence was detected on x-ray film and densitometrically scanned within the linear range using both volume and profile analyses (Bio-Rad Laboratories, Inc., model GS-700 phosphorimager).
RNA analysis
Total RNA (10 µg/sample) was resolved by electrophoresis on
1% denaturing agarose gels, and Northern blot analysis was used for
quantitation of SERCA2 and PLB messenger RNAs (mRNAs), which were
normalized to 18S ribosomal RNA. Murine PLB complementary DNA (provided
by Dr. E. G. Kranias, University of Cincinnati, Cincinnati, OH)
and rat SERCA2 complementary DNA (proved by Dr. W. H. Dillmann,
University of San Diego, San Diego, CA) were radiolabeled by the random
priming method and used to detect the corresponding mRNAs on the
Northern blots as previously described (15).
Statistical analysis
All results are expressed as the mean ± SE.
Unpaired Student’s t test was used for statistical
comparison of two groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Rats receiving
continuous infusion of T3 at 2.5 and 7.0 µg/day
had significantly elevated serum T3 levels of
116 ± 5 and 420 ± 45 ng/dl, respectively. As expected,
serum free T3 levels were also significantly
higher than control values and increased proportionally to the
T3 dose (Table 1). The mean heart rate in the
hypothyroid group [199 ± 26 beats/min (bpm)] was significantly
lower than that in controls (293 ± 6 bpm), and this was increased
significantly to 295 ± 10 and 352 ± 17 bpm with the two
treatment doses of T3, respectively. As
previously reported (12, 15), heart weight to body weight ratios were
significantly lower in the hypothyroid rats compared with the controls,
and both T3 treatment regimens significantly
increased cardiac mass, as summarized in Table 1. The mean body weight
was 261 ± 5 g, and body weights were not different among the
experimental groups.
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Cardiac function
Systolic contractile function was measured using M-mode
echocardiography. The ejection fraction (percentage) was lower in the
hypothyroid rats by 22% (P < 0.01) compared with
controls, and this measure of cardiac function was returned to normal
in the two T3-treated groups (Table 1
).
Fractional shortening was similarly decreased by 33%
(P < 0.01) in the hypothyroid hearts (data not
shown).
RNA and protein analysis of SERCA2 and PLB
Hypothyroidism produces predictable changes in the expression of a
number of T3-responsive genes in the myocardium
as previously reported (3, 12, 15, 16). The representative Western blot
in Fig. 1 shows that the PLB pentamer content
in the hypothyroid LV was increased significantly to 149% of the
control value (Table 2).
T3 treatment of the hypothyroid animals decreased
the PLB protein content toward control values with a trend that
suggests a dose responsiveness of this effect. The ventricular SERCA2
protein concentration was not significantly altered by thyroid hormone
status, although there was a trend toward lower values (10%) in the
hypothyroid heart and an increase (12%) with T3
treatment. When total LV SERCA2 (concentration x mass) was
calculated, it was observed that hypothyroidism decreased SERCA2 by
19% (P < 0.05), and T3
treatment at the 2.5 and 7.0 µg doses increased SERCA2 by 22% and
30%, respectively (P < 0.05).
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). This change in the
ratio of SERCA2/PLB protein in response to T3
treatment corresponded to a change in the ratio of their mRNA, with an
increase in SERCA2 mRNA from 0.57 ± 0.15 to 1.15 ± 0.04
(P < 0.05), measured for the 7 µg/day dosage (Table 2). These results are similar to those previously reported (1, 3).
Phospholamban phosphorylation
The phosphorylation state of PLB is an important determinant of
its inhibitory action on SERCA2 activity (4, 10). Western blot analysis
of the 10 phosphorylated PLB pentameric subunits resolved by gel
electrophoresis is shown in Fig. 2.
Consistent with the Western blot in Fig. 1, in which the individual
phosphorylated PLB pentamers were not resolved, was the increased
quantity of PLB in the hypothyroid LV samples compared with control or
T3-treated samples. Furthermore, the pattern of
phosphorylated PLB subunits was markedly altered with
T3 treatment. The quantity of nonphosphorylated
pentamer (25 kDa) in the hypothyroid LV (9.38 ± 2.29) was
significantly greater than that in either control (2.24 ± 0.48)
or T3-treated LV (1.67 ± 0.17 and 0.50
± 0.04 for 2.5 and 7.0 µg T3, respectively;
Table 3). Moreover,
T3 treatment of the hypothyroid rats
increased the phosphorylation state of the PLB pentamers, which is
indicated by a shift to the higher mol wt protein bands seen in Fig. 2.
Densitometric profile analysis of the PLB bands in the hypothyroid and
2.5 µg T3-treated samples is shown in Fig. 3, with P-0 indicating the nonphosphorylated
PLB pentamer, and P-10 corresponding to the PLB pentamer with all 10
serine/threonine sites phosphorylated. Densitometric tracings of the 7
µg/day T3 treatment group showed decreased
amounts of total PLB protein (Table 2) and a leftward shift of the
phosphorylated pentamers similar to the lower T3
dose.
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shows the quantitation of the PLB pentamers using densitometric
profile analysis and expressing the sum of peak heights of the PLB
pentamers containing 6–10 phosphates as a fraction of total PLB
pentamers (P6–P10/P0–P10). This analysis showed that a greater
proportion of the PLB pentamers was highly phosphorylated in the
control and T3-treated samples compared with the
hypothyroid LV. In addition, T3 treatment at the
highest dose (7.0 µg) increased PLB phosphorylation to values
significantly higher than PLB phosphorylation in control animals
(0.86 ± 0.04 vs. 0.72 ± 0.03, respectively;
Table 3). Treatment with T3 for 1 and 2 h did not change the phosphorylation profile of the hypothyroid heart compared with that in controls (data not shown), suggesting that the changes in PLB phosphorylation were the result not of phosphorylation mechanism of preexisting pathways but, rather, of altered kinase/phosphatase content, activity, or distribution within the myocyte.
Site-specific PLB phosphorylation
To identify potential mechanisms for the
T3-mediated increase in PLB phosphorylation,
Western blot analysis of PLB was used to determine the extent of
phosphorylation of serine 16 and threonine 17 in control, hypothyroid,
and T3-treated hearts. This was accomplished
using phosphoserine (PS-16)- and phosphothreonine (PT-17)-specific
antibodies, as shown in Fig. 4, and compared
with the monoclonal antibody A1 that recognizes all PLB subunits (14).
As the total PLB protein content in the
T3-treated samples was significantly less than
that in hypothyroid LV, the Western blot analysis was performed using
twice the amount of T3-treated sample compared
with hypothyroid sample (600 vs. 300 µg protein,
respectively). As shown in Fig. 4, the proportion of highly
phosphorylated PLB (more than five phosphates) in the
T3-treated LV was greater than that in the
hypothyroid sample, observed using the A1 antibody. In the hypothyroid
sample, the distributions of phosphoserine and phosphothreonine were
similar, except the lowest PS-16 band was not recognized by PT-17
antibody, whereas the highest phosphorylated PLB band was not
recognized by PS-16 antibody, but was recognized by PT-17 antibody. In
contrast, the phosphorylation pattern in the
T3-treated sample shows that PT-17 readily
recognizes PLB containing more than five phosphates, supporting the
hypothesis that cAMP-dependent protein kinase (PKA)-mediated
phosphorylation on serine 16 may occur in conjunction with or perhaps
as a prerequisite for calcium/calmodulin-dependent kinase
phosphorylation of threonine 17 as a result of T3
treatment (9, 14).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The current data support previous observations in which T3 treatment of the hypothyroid heart increased the SERCA2/PLB ratio by significantly decreasing PLB protein (3, 18). These changes varied with the treatment dose of T3, but the results did not reach statistical significance. Although T3 did not alter the SERCA2 protein concentration (per mg total protein), it significantly increased the total content of SERCA2 protein and mRNA in the LV, consistent with prior reports of thyroid hormone effects on cardiac SERCA2 gene expression and on altered mRNA translation efficiency (4, 12, 19).
The present finding that the phosphorylation state of the individual PLB pentamers was altered with hypothyroidism and responded to T3 treatment is novel. Kiss et al. (3) previously reported that when isoproterenol-stimulated phosphate incorporation into phospholamban was measured between hearts of different thyroid states, there was increased phosphorylation in the hypothyroid hearts compared with those from euthyroid and T3-treated animals. This can be interpreted as demonstrating a decrease in baseline phosphorylation of the hypothyroid hearts as well as an increase in total PLB protein content and is consistent with our current observations. Additionally, the current data indicate that the T3-mediated change in PLB phosphate content produced a shift in distribution of phosphorylated PLB pentamers to those containing greater than five phosphates. Unlike the well described effects of thyroid hormone on PLB gene transcription (3), the current findings point to a posttranscriptional, covalent modification of the PLB molecule. These effects would be expected to increase the diastolic rate of relaxation (3, 20) and to enhance the systolic contractility as we observed in the current experiments. Whether the trend of increased PLB phosphorylation with the higher T3 dose could result in enhanced myocyte relaxation remains to be determined.
Phosphorylation of PLB after ß-adrenergic stimulation occurs primarily by PKA phosphorylation of serine 16 (9, 20). Many of the effects of thyroid hormone on the heart appear to be synergistic with ß-adrenergic receptor stimulation (21). T3 treatment may enhance cAMP production in the myocyte, leading to increased PKA activity and increased phosphorylation of PLB (5, 22, 23). Kaasik et al. (24) suggested that the PKA pathway may be responsible for PLB phosphorylation and thyroid hormone-mediated changes in atrial contractility and chronotropy. However, we did not observe a change in PLB phosphorylation after acute T3 treatment and conclude that if T3 alters PKA activity, then it does so through a classic genomic mechanism (1, 12).
Phosphorylation of PLB also occurs on threonine 17 by a calcium/calmodulin-dependent protein kinase (21). As T3 can simultaneously augment calcium cycling in cardiac myocytes (3, 6, 17), this could explain our observation of an increase in threonine 17 phosphorylation. In the present study the shift in phosphorylated PLB pentamers, with increases in both serine 16 and threonine 17, is consistent with multiple sites of regulation. The observation that acute T3 treatment did not alter the PLB phosphorylation profile suggests that the mechanism may involve changes in the protein phosphatase content, activity, or distribution within the myocyte (25).
The present studies confirm prior observations that the positive inotropic effect of thyroid hormone is mediated through transcriptional level regulation of PLB and SERCA2 and extend those findings by identifying a novel pathway by which PLB phosphorylation can be regulated. We further conclude that protein phosphorylation/dephosphorylation cascades may underlie the cellular actions of thyroid hormone on the heart.
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Footnotes |
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Received January 7, 2000.
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