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Transgenic overexpression of the sarcoplasmic reticulum Ca²⁺ATPase improves reticular Ca²⁺ handling in normal and diabetic rat hearts¹

ROLAND VETTER², UWE REHFELD, CHRISTOPH REISSFELDER, WOLFGANG WEISS, KAY-DIETRICH WAGNER^*, JOACHIM GÜNTHER^*, ANNETTE HAMMES, CARSTEN TSCHÖPE^#, WOLFGANG DILLMANN and MARTIN PAUL

Department of Toxicology, Institute of Clinical Pharmacology and Toxicology and
^# Department of Cardiology and Pulmonology, Benjamin Franklin Medical Center, Freie Universität Berlin, D-14195 Berlin, Germany;
^* Department of Physiology, Humboldt University-Charité, D-10117 Berlin, Germany;
Max Delbrück Center for Molecular Medicine, D-13122 Berlin-Buch, Germany; and
Department of Medicine, University of California, San Diego, La Jolla, California, USA

²Correspondence: Department of Toxicology, Institute of Clinical Pharmacology and Toxicology, Benjamin Franklin Medical Center, Freie Universität Berlin, D-14195 Berlin (Dahlem), Germany. E-mail: rvetter{at}zedat.fu-berlin.de

SPECIFIC AIMS

Slowed relaxation in diabetic cardiomyopathy is partially related to diminished expression of the sarcoplasmic reticulum (SR) Ca²⁺ ATPase SERCA2a. To evaluate the effect of SERCA2a overexpression on SR Ca²⁺ handling in diabetic cardiomyopathy, we 1) generated SERCA2 transgenic rats (SERCA2-TGR) harboring a human cytomegalovirus enhancer/chicken ß-actin promotor-controlled rat SERCA2 transgene, 2) characterized their cardiac SR and contractile function, and 3) examined whether transgene expression may rescue SR Ca²⁺ transport in streptozotocin(STZ)-induced diabetes.

PRINCIPAL FINDINGS

1. Cardiac phenotype of the novel SERCA2 transgenic rat model
Southern blot analysis of rat genomic tail DNA with a transgene-specific probe identified four separate female and one male founder rats. Two of these gave rise to lines L1375 and L1167. Heterozygous male rats of the latter line were mainly used in this study. Heart and body weight characteristics of these SERCA2-TGR did not differ from age- and sex-matched wild-type (WT) controls. Northern blot analysis revealed increased steady-state SERCA2 mRNA levels in all chambers of the heart of transgenic animals. Quantitation of the normalized left ventricular mRNA coding for SERCA2 indicated a 1.5 ± 0.18-fold (P<0.05) increase in SERCA2-TGR of line 1167. Increased SERCA2 message was observed in other tissues such as skeletal muscle, diaphragm, esophagus, and kidney. To examine whether increased SERCA2 message was translated into increased cardiac SR Ca²⁺ ATPase protein Western blot analysis was performed using a SERCA2-specific antiserum. The SERCA2 protein level was increased by 26% in transgenic myocardium of line L1167 (P<0.05). The level of phospholamban, a modulatory protein of the cardiac SR Ca²⁺ ATPase SERCA2a, was not altered in transgenic rats. Moderate overexpression of SR Ca²⁺ ATPase protein in SERCA2-TGR resulted in a marked increase in SR Ca²⁺ transport activity as determined by measurements of oxalate-supported SR Ca²⁺ uptake in left ventricular homogenates. As shown in Fig. 1 B for different free Ca²⁺ concentrations the rate of in vitro Ca²⁺ uptake was significantly higher in SERCA2 overexpressors. The maximum uptake rate V_max of line L1167 was increased by 37% (P<0.05 vs. respective WT controls). A comparison of the linear dependencies in Fig. 1A revealed a significant increase in the rate of cardiac Ca²⁺ uptake for a second transgenic line (L1375) by 40% (P<0.05 vs. WT controls).

View larger version (18K): [in this window] [in a new window]   Figure 1. Differences in sarcoplasmic reticular Ca2+ uptake in left ventricular homogenates prepared from adult SERCA2 transgenic and age-matched wild-type (WT) control rats. A) Time dependence of oxalate-supported Ca2+ uptake in homogenates from homozygous SERCA2 transgenic rats (n=4) and WT controls (n=3) of line L1375. Concentration of free Ca2+ was 0.21 µM. B) Rates of oxalate-supported Ca2+ uptake in homogenates from heterozygous SERCA2 transgenic rats (n=5) and WT controls (n=5) of line L1167 at different concentrations of free Ca2+. Calculated maximum rate values were 25.4 ± 2.1 and 18.5 ± 1.6 nmol Ca2+/mg protein/min in transgenic and WT rats, respectively. Values are means ± SE. *P < 0.05 vs. respective WT control values.

2. Improved cardiac contractile properties of SERCA2 transgenic rats
To further study whether the increased expression of functional SERCA2 protein resulted in altered contractility, experiments with isolated papillary muscles were performed. Compared with papillary muscles of WT rats, a heterozygous line L1167 exhibited increased contraction amplitude (+48%) and rates of contraction (+34%) and relaxation (+35%) (P<0.05 vs. WT, respectively). To enforce the SR of cardiomyocytes to handle increased amounts of intracellular Ca²⁺ during the contraction-relaxation cycle, the papillary muscleswere exposed to increasing concentrations of the positive inotropic agent forskolin. The latter is known to cause a tremendous increase in intracellular Ca²⁺ due to stimulation of Ca²⁺ influx via L-type Ca²⁺ channels. The cardiotonic drug produced typical positive inotropic responses and accelerated dose-dependently the relaxation in both transgenic and control muscles. However, these effects were even more expressed in papillary muscles with transgenic SERCA2 overexpression. For example, the relaxation index dF/dt_min/PF was 1.3-fold higher in transgenic papillary muscles exposed to 10 µmol/L forskolin if compared with control conditions in the absence of the drug. The increase of this index in response to the same dose of forskolin was < 10% in WT controls. Stronger inotropic effects than in WT controls also paralleled the more intense lusitropic responses of papillary muscles of SERCA2-TGR. This indicates indirectly a higher Ca²⁺ loading of the cardiac SR of SERCA2-TGR under conditions of enhanced Ca²⁺ cycling compared with WT controls.

3. Rescued cardiac SR Ca²⁺ transport in SERCA2 transgenic rats with STZ-induced diabetes
Compared with nondiabetic WT papillary muscles (PM), contractility and rate values of relaxation were significantly depressed in diabetic WT but not in diabetic SERCA2-TGR. The contractile responses of PM to ß-adrenergic stimulation and extrasystolic test stimuli (ES) were markedly depressed in diabetic WT rats compared with nondiabetic WT controls. This depression was either abolished or occurred only partially in diabetic transgenic rats overexpressing SERCA2a. In particular, hastening of relaxation with increasing concentrations of isoprenaline was similar in diabetic SERCA2-TGR and nondiabetic WT rats but was markedly reduced in papillary muscles of diabetic WT rats. This was associated with an attenuated depression of the postextrasystolic responses to ES compared with diabetic WT rats. As these special responses are thought to reflect differences in the Ca²⁺ recycling activity of the SR in situ, we further examined in vitro whether functional overexpression of SERCA2 can counteract the depression of SR Ca²⁺ transport function in diabetic cardiomyopathy. We analyzed homogenate SR Ca²⁺ uptake and immunoreactive SERCA2 protein levels in transgenic rat hearts 6 wk after induction of diabetes mellitus by a single dose of 65 mg STZ/kg body wt. In diabetic WT control rats, rate of Ca²⁺ uptake measured at 0.34 µmol/L free Ca²⁺ was decreased by 52%. Comparison of transgenic and WT rats with diabetes revealed 35% higher Ca²⁺ uptake rate values in SERCA2-TGR (P<0.05). Similar differences were observed at saturating free Ca²⁺ concentrations (Fig. 2 ),when possible modulatory influences of phospholamban on the SR Ca²⁺ ATPase can be discounted. These alterations in SR Ca²⁺ uptake were linked to similar changes in SERCA2 protein. Left ventricular SERCA2 protein level was significantly decreased by 33% in diabetic WT rats (P<0.05 vs. nondiabetic WT). This decline was attenuated in diabetic SERCA2-TGR. Compared with diabetic WT rats, the SERCA2 protein level was 25% higher in diabetic SERCA2-TGR. Abundance of phospholamban was not altered. Furthermore, a linear relationship (r=0.77, P<0.001) was observed between rate values of SR Ca²⁺ uptake and SERCA2 protein level if the respective numbers of nondiabetic WT rats, diabetic WT rats, and diabetic SERCA2-TGR were plotted together (Fig. 2) . Thus, support for rescued cardiac SR Ca²⁺ recycling in experimental diabetes by transgenic SERCA2 overexpression was obtained by both in situ experiments with isolated PM and in vitro measurements of SR Ca²⁺ uptake and SERCA2 protein in cardiac homogenates.

View larger version (20K): [in this window] [in a new window]   Figure 2. Relationship between sarcoplasmic reticulum Ca2+ uptake rate values and immunoreactive SERCA2a protein levels in left ventricular homogenates of nondiabetic wild-type (WT) control rats, diabetic WT rats, and diabetic SERCA2 transgenic rats (TGR) of line L1167. Values of SR Ca2+ uptake are means of duplicate determinations at a free Ca2+ concentration of 2.68 µM. Uptake reaction was terminated after 2 min. For Western blotting, homogenate protein was 10 µg/lane. Regression equation: y = 1.225 x +37.507.

CONCLUSIONS AND SIGNIFICANCE

This is the first report of a novel transgenic rat model with cardiac SERCA2a overexpression. The model was used to target the SR Ca²⁺ ATPase expression to rescue SR Ca²⁺ handling in diabetic cardiomyopathy. It is well known that slowed relaxation is a prominent feature of impaired cardiac performance in diabetic cardiomyopathy. In the STZ diabetic model, it is mediated in part through depressed uptake of Ca²⁺ by the cardiac SR. A decreased expression of SERCA2a, leading to reduced abundance of Ca²⁺-pumps in the SR membrane, appears to be one of the contributing mechanisms. In this study, we 1) corroborate these previous findings for 6 wk STZ diabetic WT rats and 2) demonstrate that an intervention directed specifically at SERCA2a expression, without influencing the overall metabolic status, is useful to improve the compromised SR Ca²⁺ handling in the diabetic heart. This was associated with a partial restoration of cardiac relaxation to normal. Specifically, our results provide the first experimental evidence for a SERCA2 transgene-mediated improvement of the Ca²⁺ transporting function of the cardiac SR in a diabetic rat model. They therefore extend current knowledge on the role of altered SR Ca²⁺ ATPase gene expression in the diabetic heart. The results also suggest that targeting SERCA2 expression might constitute a future potential therapeutic approach to rescue cardiac SR Ca²⁺ handling and contractile dysfunction in diabetes. Our findings are generally in line with recently published reports on specifically targeting gene expression of the SR Ca²⁺ ATPase or its activity modulator, phospholamban, in murine models to rescue SR Ca²⁺ handling and contractile performance in diseased states other than diabetic cardiomyopathy.

View larger version (38K): [in this window] [in a new window]   Figure 3. Schematic diagram showing differences in cardiac Ca2+ pump abundance linked to altered reticular uptake of Ca2+ in membranes (gray rings) of the cardiac sarcoplasmic reticulum (SR) of wild-type and SERCA2 transgenic rats (SERCA2-TGR) without (to the left) and with (to the right) streptozotocin-induced diabetic cardiomyopathy. The small circles indicate Ca2+ pump molecules encoded either by the endogenous SERCA2 gene (empty circles) or the SERCA2 transgene (filled circles). Arrows specify the SERCA2-catalyzed uptake of Ca2+ from the cytosol into the lumen of the SR. Differences in the amount of Ca2+ taken up by the SR are illustrated by different font sizes for Ca2+. Note: The number of functional Ca2+ pumps is higher in SERCA2-TGR due to expression of the SERCA2 transgene. In diabetic cardiomyopathy, this compensates partially for loss of Ca2+ pump molecules encoded by the endogenous SERCA2 gene and results in partial restoration of defective SR Ca2+ transport and relaxation to normal.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-1019fje; to cite this article, use FASEB J. (August 19, 2002) 10.1096/fj.01-1019fje

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