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Gender differences in myosin heavy chain- and phosphorylated phospholamban in diabetic rat hearts

¹Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Cincinnati 45267-0575; and ²Department of Oral Biology, Ohio State University, Columbus, Ohio 43210-1267

Submitted 12 June 2003 ; accepted in final form 23 July 2003

	ABSTRACT

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The objective of this study was to determine whether a gender difference exists in myosin heavy chain (MHC) isoform or sarcoplasmic reticulum protein levels in diabetic rat hearts. As is the case with normal rodent hearts, all four chambers of the control rat hearts expressed almost 100% MHC-. In 6-wk diabetic rats, MHC- expression in ventricles of males was significantly greater (78 ± 7%) than in females (50 ± 5%). The cardiac sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a) protein level was decreased and the phospholamban (PLB) protein level was increased in the left ventricle of diabetic rats, but there was no difference between male and female diabetic rats. The phosphorylated PLB level was decreased more in male than in female diabetic rats. Insulin treatment completely normalized blood glucose level, cardiac SERCA2a and PLB protein levels, and the decrease in MHC- levels in both male and female diabetic rats. Insulin treatment completely normalized serum insulin and almost completely normalized phosphorylation of PLB at serine 16 in male diabetic rats. Although insulin treatment completely normalized serum insulin levels in male diabetic rats, in females it only partially normalized serum insulin levels. Also, insulin treatment almost completely normalized phosphorylation of PLB at threonine 17 in female diabetic rats; however, the increase was significantly greater than that identified for insulin-treated male diabetic rats. We conclude that higher levels of MHC- and dephosphorylated PLB may contribute to more contractile dysfunction in male than in female diabetic rat hearts, and that phosphorylation of PLB at threonine 17 is more responsive to insulin in female diabetic rat hearts.

diabetes; cardiomyopathy; sarcoplasmic reticulum; calcium ATPase

Expression of the myosin heavy chain (MHC)- isoform is linked to depressed cardiac contraction (18, 22). Gender differences in MHC- expression in rat hearts have been reported (12, 23). Expression of MHC- has been demonstrated in male streptozotocin-induced diabetic rat hearts (7, 8, 15). Greater MHC- expression may contribute to more severely depressed contraction in male than female diabetic rat hearts. However, this premise has not been examined in streptozotocin-induced diabetic rats.

Overexpression of phospholamban (PLB) has been linked to depressed cardiac contraction (13). Gender differences in phosphorylated PLB have also been reported (6). Recently, we and others have demonstrated overexpression of dephosphorylated PLB levels in male streptozotocin-induced diabetic rat hearts (4, 14, 26). More dephosphorylated PLB may also contribute to more depressed contraction in male than female streptozotocin-induced diabetic rat hearts. This premise also remains unverified.

MHC and PLB have pivotal roles in regulation of the kinetics of contraction and relaxation, respectively, in mammalian hearts. The objective of this study was to examine the hypothesis that there is more MHC- and/or dephosphorylated PLB in male than female diabetic rat hearts. The rationale of the study is that more MHC- and dephosphorylated PLB are likely to form the molecular basis for more depressed contraction in male than female diabetic rat hearts.

	METHODS

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Animals. Six 7-wk-old (140–150 g body wt) male and female Wistar rats were made diabetic with a single injection of streptozotocin (65 mg/kg) into a tail vein (10). The age-matched control rats were injected with 0.1 ml of vehicle (0.1 M sodium citrate, pH 4.5). Food and water were available ad libitum to all rats. Four weeks after the injection of streptozotocin, a randomly selected group of diabetic rats was treated daily at 6:00 PM with a subcutaneous injection of 5 IU of PZI (cow and pig) insulin (Blue Ridge Pharmaceuticals; Greensboro, NC) for 2 wk. All control, diabetic, and insulin-treated diabetic rats were killed 6 wk after the streptozotocin injection. Blood glucose levels were determined using a glucometer (Bayer; Elkhart, IN), and serum insulin levels were measured using a radioimmunoassay kit (Amersham Life Sciences; Little Chalfont, UK). Serum-free T₃ levels were measured using a radioimmunoassay kit (Diagnostic Products; Los Angeles, CA).

The use of rats in this study conformed to the Guide for the Care and Use of Laboratory Animals published by National Institutes of Health [DHEW Publication No. (NIH) 85-23, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205], and experiments were executed according to a protocol approved by the University of Cincinnati Institutional Animal Care and Use Committee.

Collection of cardiac tissue samples. Rats were anesthetized with pentobarbital sodium (60 mg/kg) at 9:00 AM. After 30 min of deep sleep, each rat was killed and the heart was excised and immediately placed in ice-cold saline. The right and left atria and right and left ventricles were separated over an ice bed. The right and left ventricles were either kept together or, in some experiments, the left ventricular free wall at median level was transmurally subsectioned into epi-, mid-, and endocardia. The samples were frozen in liquid nitrogen and stored at –80°C until analysis of MHC and sarcoplasmic reticulum (SR) proteins.

Gel sample preparation for MHC isoform analysis. Samples were prepared for gel electrophoresis according to the methods of Blough et al. (1). Each sample (20–30 mg of tissue) was placed in a microcentrifuge tube, and sample buffer was added (30 µl/mg tissue). The sample was then homogenized for 10 s, heated for 2 min at 95°C, chilled on ice for 5 min, and finally centrifuged. The supernatant was saved and diluted 1:10 with sample buffer. The diluted samples (2 µl of ventricle or 3 µl of atrium) were loaded for electrophoresis.

Gel electrophoresis. The gel preparation methods and running conditions were identical to those described by Reiser and Kline (19). Gels were run at 8°C in an SE 600 unit (Hoefer Scientific; San Francisco, CA). Stacking gels consisted of 4% acrylamide (50:1 acrylamide-to-bis-acrylamide ratio) and 5% (vol/vol) glycerol (pH 6.8). Separating gels consisted of 7% acrylamide (50:1 acrylamide-to-bis-acrylamide ratio) and 5% (vol/vol) glycerol (pH 8.8). Gels were run at constant 200 V for 24 h, fixed for 1 h in a solution that contained 50% (vol/vol) ethanol and 10% (vol/vol) acetic acid, and then further fixed for a minimum of 2 h in 5% glutaraldehyde. Gels were then silver stained and scanned with a GS 300 densitometer (Hoefer Scientific) to determine the amount of MHC- or - present relative to the total MHC in each sample. The sample loads were within the linear range of the relationship between the amount of sample loaded and the densitometric peak area (19).

Measurements of SR protein and phosphorylated PLB levels. The quantitative immunoblot technique was used to determine sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a), total PLB, and PLB phosphorylated at serine 16 (PLB-P^ser16) and threonine 17 (PLB-P^thr17) levels (26).

Statistical analysis. ANOVA and t-tests were employed to evaluate differences between groups. A change was considered significant when P < 0.05. Values are expressed as means ± SE of at least four rats in each group.

	RESULTS

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Characteristics of experimental animals. The non-fasting blood glucose levels were approximately three-fold higher in male and female streptozotocin-treated rats at the time they were killed (Fig. 1). The serum insulin levels in male and female diabetic rats were 15% of the respective control rats. There were no statistically significant differences in blood glucose or serum insulin levels between male and female diabetic rats. Serum-free T₃ levels were decreased by 66% in male and 78% in female diabetic rats. The difference between male and female diabetic rats was not statistically significant. These data demonstrate that there was no significant gender difference in the severity of hyperglycemia, hypoinsulinemia, or hypothyroidism in diabetic rats.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Blood glucose (A), serum insulin (B), and free T3 (C) levels in experimental rats. Rats were 6-wk diabetic and age-matched controls. Insulin-treated diabetic rats were 4-wk diabetic that were afterward given 2 wk of daily insulin treatment as described in METHODS. MC, male control; MD, male diabetic; MI, male insulin-treated diabetic; FC, female control; FD, female diabetic; FI, female insulin-treated diabetic. Results are means ± SE; n = 5 rats; *P < 0.05 vs. control group; #P < 0.05 vs. diabetic group.

Insulin treatment almost completely normalized blood glucose levels in both male and female diabetic rats (Fig. 1). The serum insulin levels of insulin-treated male diabetic rats, which were measured 15 h after the insulin injections, were similar to those of male control rats. On the other hand, the insulin levels of insulin-treated female diabetic rats remained significantly below the levels of female control rats. Although insulin treatment completely normalized serum-free T₃ levels in insulin-treated male diabetic rats, these levels remained below normal in insulin-treated female diabetic rats.

MHC isoform composition. Almost 100% of the total MHC protein in the atria and ventricles of control rats was the MHC- isoform (Fig. 2). The relative level of MHC- increased in both ventricles of the diabetic rats, and there was a linear correlation in the expression of MHC- between right and left ventricles of diabetic rats. The ventricular expression of MHC- in male 6-wk diabetic rats was 78 ± 7% compared to 50 ± 5% (P < 0.05) in female 6-wk diabetic rats (Fig. 3). There was no detectable MHC- expression in the atria of diabetic rats (data not shown). Insulin treatment of diabetic rats for 2 wk reduced MHC- levels to 19 ± 1% in males and 18 ± 2% in females. Insulin treatment was equally effective in reducing MHC- expression in male and female diabetic rats.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Myosin heavy chain (MHC) isoform levels in left and right ventricles (LV and RV, respectively) of male control and diabetic rats compared with a duration of diabetes (A and B) of 2, 4, or 6 wk (2W, 4W, and 6W, respectively). Data are means ± SE; n = 5 rats. Correlation of MHC- expression between LV and RV of male diabetic rats is shown (C); data points represent values of 2-, 4-, and 6-wk male diabetic rats. CLV, control LV; DLV, diabetic LV; CRV, control RV; DRV, diabetic RV.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Gender differences in MHC- levels in diabetic rats: an example of relative MHC- and - levels in 6-wk male and female diabetic insulin-treated and respective age-matched control rats (top). Means ± SE of 5 rats are shown for each group (bottom). *P < 0.05 vs. control group; #P < 0.05 vs. diabetic group.

SR protein levels. In 6-wk diabetic rats, the total SERCA2a protein level was reduced by 28% in males and by 39% in females compared with respective control rats (Fig. 4A). The difference in SERCA2a expression between male and female diabetic rats was not statistically significant. Total PLB protein level was increased by 47 and 45%, respectively, in male and female diabetic rats (Fig. 4B). These data suggest that there were no significant gender differences in the decrease in SERCA2a and the increase in PLB total protein levels in these diabetic rats.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a; A) and total phospholamban (PLB; B) protein levels in 6-wk male and female diabetic rats, insulin-treated diabetic rats, and age-matched control rats. Representative quantitative immunoblots are shown (top). Values are means ± SE of 5 rats in each group. *P < 0.05 vs. control group.

Phosphorylated PLB protein levels. The levels of PLB-P^ser16 and PLB-P^thr17 in control 6-wk male diabetic rats were more than in control female rats. In 6-wk diabetic rats, the levels of PLB-P^ser16 were decreased by 46% in males and 26% in females compared with respective control rats (Fig. 5A). The levels of PLB-P^thr17 were decreased by 56% in 6-wk male diabetic rats and by 26% in females (Fig. 5B). This difference in the phosphorylated PLB level between male and female diabetic rats was statistically significant (P < 0.05). Insulin treatment almost completely normalized the decreased PLB-P^ser16 levels in male and female diabetic rat hearts. Insulin treatment also almost completely normalized the decreased PLB-P^thr17 levels in male diabetic rat hearts. However, insulin treatment resulted in more PLB-P^thr17 in female than in male insulin-treated diabetic rats. These data suggest that there is more dephosphorylated PLB in male than in female diabetic rat hearts and that insulin treatment increases PLB-P^thr17 levels more in female than in male diabetic rat hearts.

View larger version (41K): [in this window] [in a new window]   Fig. 5. Gender differences in PLB phosphorylated at serine 16 (PLB-Pser16; A) and at threonine 17 (PLB-Pthr17; B) in diabetic rats. Representative quantitative immunoblots are shown (top). Values are means ± SE of 5 rats in each group. *P < 0.05 vs. control group; #P < 0.05 vs. diabetic group; **P < 0.05 vs. male insulin-treated diabetic rats.

	DISCUSSION

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We report the novel findings that male diabetic rat hearts have greater levels of MHC- and dephosphorylated PLB than female diabetic rat hearts. The results provide a molecular basis for the more severe decrease in the rate of left ventricular muscle contraction and relaxation that is reported in streptozotocin-induced male vs. female diabetic rats (2, 3, 20).

We also report here a novel finding of less serum insulin and free T₃ levels in insulin-treated female diabetic rats than in male diabetic rats. The difference in T₃ levels could be related to the decrease in insulin levels. The difference in insulin levels did not produce a difference in blood glucose levels between insulin-treated male and female diabetic rats. The reason for the difference in serum insulin levels between insulin-treated male and female diabetic rats is unclear. However, this difference is unlikely to be responsible for the more depressed contraction and relaxation rates that are observed in male diabetic rat hearts, as there were no significant differences in blood glucose and serum insulin levels between male and female diabetic rats.

Rodent hearts predominantly express MHC- except in certain pathological conditions when expression of MHC- occurs concomitant with a decrease in MHC-. Recently, it was demonstrated that targeted expression of even 12% MHC- in adult mouse hearts causes a significant (19%) decrease in the rate of systolic contraction (21). In the present study, we observed 30% more MHC- in male than female diabetic rat hearts. This higher level of MHC- is likely to contribute to the more depressed contraction observed in male diabetic rat hearts, because expression of an even smaller level has been shown to depress the rate of cardiac contraction in mouse hearts (21). Thus higher levels of MHC- may have contributed to the more depressed rate of contraction observed in male vs. female diabetic rat hearts (2, 3, 20). The mechanism that underlies the gender difference in MHC- expression in diabetic rat hearts is unclear. Hypothyroidism has been shown to promote MHC- expression (16). We observed no significant difference in serum T₃ levels between male and female diabetic rats. Rodrigues and McNeill (20) reported no significant difference in serum T₄ levels between male and female 6-wk streptozotocin-induced diabetic rats. Thus low thyroid hormone levels could not account for the gender difference in MHC- expression in diabetic rats. Differential alteration of transcription of the MHC- gene by a factor other than thyroid hormone receptor signaling is probably responsible for the greater MHC- protein level in male diabetic rats. Thus the mechanism that underlies the greater MHC- expression in male vs. female diabetic rat hearts remains to be determined.

Targeted overexpression of PLB in mouse hearts has been shown to decrease the affinity of SERCA2a for Ca²⁺ and cause depression of contraction and relaxation (13). We have shown previously that increased total PLB and decreased phosphorylated PLB protein levels significantly decrease the affinity of SERCA2a for Ca²⁺ (4, 26). In the present study, we observed similar increases in total PLB in male and female diabetic rats. However, the fraction of PLB that was phosphorylated was lower in male than in female diabetic rat hearts. Higher levels of dephosphorylated PLB in male diabetic rat hearts is likely to lower the affinity of SERCA2a for Ca²⁺ more than in female diabetic rat hearts. This is likely to decrease SERCA2a activity and cytosolic Ca²⁺ cycling kinetics more in male than in female diabetic rat hearts. Thus more dephosphorylated PLB is likely to contribute to more depressed rates of relaxation and contraction as observed in streptozotocin-induced male diabetic rat hearts (2, 3, 20). We have also observed more PLB-P^thr17 in insulin-treated female than male diabetic rats. This implies that in female diabetic rat hearts, phosphorylation of PLB at threonine-17 is more responsive to insulin treatment. The mechanism that underlies these gender differences in PLB phosphorylation is unclear. Phosphorylation of PLB at serine-16 by protein kinase A (PKA) and at threonine-17 by Ca²⁺ calmodulin kinase II is one of the end points of -adrenergic receptor signaling (5). PKA has been shown to increase and Ca²⁺ calmodulin kinase II remained unchanged in streptozotocin-induced diabetic rat hearts (17). On the other hand, impaired contractile response to -adrenergic receptor stimulation has been observed in streptozotocin-induced male diabetic rat hearts (11, 25). Gender-specific alterations of a step in the -adrenergic receptor signaling pathway in diabetic rat hearts could be responsible for the differences in PLB phosphorylation.

In summary, the results of this study reveal greater levels of MHC- and dephosphorylated PLB in male streptozotocin-induced diabetic rat hearts. These differences individually and independently are likely to contribute to more depressed contraction in male than female diabetic rat hearts. The differences in the levels of MHC- and dephosphorylated PLB provide a molecular basis for the more severely depressed rates of contraction and relaxation in male diabetic rat hearts in this model of Type 1 diabetes. However, the mechanism that underlies these changes remains to be elucidated.

	DISCLOSURES

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This study was supported by the American Diabetes Association and National Heart, Lung, and Blood Institute Grant RO1 HL-56782 (to M. A. Matlib) and Grant-In-Aid 96009610 from the American Heart Association National Center (to P. J. Reiser).

	ACKNOWLEDGMENTS

 
The authors thank Ashley Mattingly for critical reading of the manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. A. Matlib, Dept. of Pharmacology and Cell Biophysics, Univ. of Cincinnati College of Medicine, 231 Albert B. Sabin Way, PO Box 670575, Cincinnati, OH 43267-0575 (E-mail: matlibma{at}email.uc.edu).

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.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 285/6/H2688    most recent 00547.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (11) Citing Articles via Google Scholar Google Scholar Articles by Zhong, Y. Articles by Matlib, M. A. Search for Related Content PubMed PubMed Citation Articles by Zhong, Y. Articles by Matlib, M. A.