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Improvement of defective sarcoplasmic reticulum Ca²⁺ transport in diabetic heart of transgenic rats expressing the human kallikrein-1 gene

CARSTEN TSCHÖPE^*,1, FRANK SPILLMANN^*, UWE REHFELD, MATTHIAS KOCH^*, DIRK WESTERMANN^*, CHRISTINE ALTMANN^*, ANDREAS DENDORFER, THOMAS WALTHER^*, MICHAEL BADER, MARTIN PAUL, HEINZ-PETER SCHULTHEISS^* and ROLAND VETTER

^* Department of Cardiology and Pneumology,
Institute of Clinical Pharmacology and Toxicology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin;
Max Delbrück Center for Molecular Medicine, Berlin; Germany; and
Institute of Experimental and Clinical Pharmacology and Toxicology, Medical University of Lübeck, Lübeck, Germany

¹Correspondence: Department of Cardiology and Pneumology, Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12220 Berlin, Germany. E-mail: ctschoepe{at}yahoo.com

SPECIFIC AIMS

Transgenic cardiac expression of the bradykinin (BK) -forming enzyme human tissue kallikrein 1 (hKLK1) in rats is known to attenuate the development of cardiac fibrosis and left ventricular dysfunction in diabetic cardiomyopathy. To examine whether improved diastolic and systolic performance in diabetic hKLK1-expressing hearts may be due to rescued sarcoplasmic reticulum (SR) Ca²⁺ handling, we studied left ventricular (LV) function and Ca²⁺-ATPase(SERCA2a)-catalyzed SR Ca²⁺ transport after induction of streptozotocin (STZ) -induced diabetes mellitus in transgenic rats (TGR) expressing the hKLK1 gene.

PRINCIPAL FINDINGS

1. Improved left ventricular performance in diabetic hKLK1-expressing transgenic rat
There were no adverse or beneficial effects of tissue hKLK1 gene expression in transgenic rats under euglycemic conditions, which had increased basal levels of coronary bradykinin outflow and cardiac bradykinin receptor mRNA expression compared with wild-type rats (WT). LV function characteristics were similar in nondiabetic WT and TGR(hKLK1). Six weeks after a single STZ injection, both diabetic WT and TGR(hKLK1) displayed severe hyperglycemia and a marked reduction of body and wet heart weights. Analyses of LV function in anesthetized, intubated and ventilated open-chest animals using a Millar tip catheter system revealed a marked LV dysfunction in diabetic WT. LV pressure (LVP) and the maximum rate of LV pressure rise (dP/dt_max) reached only 58 and 44%, respectively, of the nondiabetic WT values. This was paralleled by a significantly slowed early LV diastolic pressure decline as indicated by altered values of maximum pressure decay dP/dt_min (–58%). The time constant of isovolumic pressure decline (tau) and pressure half-time (PHT) were increased by 43% each compared with nondiabetic WT. In diabetic TGR(hKLK1), LV dysfunction was attenuated. The LVP, dP/dt_max, and dP/dt_min values were increased by 34, 47, and 66%, respectively compared with diabetic WT (P<0.05). Furthermore, kinetic of early LV relaxation of diabetic TGR(hKLK1), which is mainly controlled by SR Ca²⁺ reuptake and characterized by the time constant tau and PHT, did not differ significantly from that of nondiabetic WT. Chronic treatment of diabetic TGR(hKLK1) with the bradykinin B₂ receptor antagonist Hoe 140 (icatibant; 500 µg·kg^–1·day^–1 s.c.) led to a significant attenuation of the transgene-related improvement of the diastolic dysfunction. Thus, targeting the cardiac KLK expression was beneficial for defective systolic and diastolic performance in diabetic cardiomyopathy; a B₂ receptor-mediated mechanism appears to be involved.

2. Improvement of defective cardiac SR Ca²⁺ transport in diabetic hKLK1 transgenic rats
To examine whether slowed relaxation in diabetic hearts was linked to altered SR Ca²⁺ transport, the rates of oxalate-facilitated ⁴⁵Ca²⁺ uptake into SR vesicles of LV homogenates were determined. Figure 1 A, B shows the dependency of SR Ca²⁺ uptake for the experimental groups. In diabetic WT, rate values of Ca²⁺ uptake were markedly decreased over a broad range of Ca²⁺ concentrations vs. nondiabetic WT (Fig. 1A ). The respective V_max value of Ca²⁺ uptake was 61% of that in nondiabetic WT. By contrast, diabetic TGR(hKLK1) exhibited no significant decrease in Ca²⁺ uptake compared with nondiabetic TGR (Fig. 1B ). Moreover, the V_max value of diabetic TGR was 42% higher compared with diabetic WT. This protection was abolished if diabetic TGR were chronically treated with Hoe 140 (Fig. 1B ), indicating involvement of a B₂ receptor-mediated mechanism. Kinetic analysis of Ca²⁺ uptake revealed no significant differences in the EC₅₀(Ca²⁺) values between the groups.

View larger version (20K): [in this window] [in a new window]   Figure 1. Differences in sarcoplasmic reticular Ca2+ uptake in left ventricular homogenates prepared from A) nondiabetic and diabetic wild-type rats (WT) as well as from B) nondiabetic and diabetic (hKLK1) transgenic rats (TGR). B) Influence of Hoe 140 treatment on Ca2+ uptake of diabetic (hKLK1) transgenic rats is shown. Ca2+ uptake was measured for 2 min in the presence of varying submicromolar free Ca2+ concentrations. Values are means ± SE (n=6–8 each).

3. Status of phospholamban phosphoylation in diabetic hKLK1 transgenic rats
Irrespective of the similarity observed in EC₅₀(Ca²⁺) values in the experimental groups, improved SERCA2a-catalyzed SR Ca²⁺ uptake in diabetic TGR(hKLK1) was linked to increased phosphorylation of phospholamban (PLB) at the Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) specific residue Thr¹⁷. Diabetic TGR(hKLK1) exhibited a 2.2-fold increase in PThr¹⁷-PLB compared with diabetic WT (Fig. 2 ). This difference was completely abolished by Hoe 140 treatment (Fig. 2) . Furthermore, a linear relationship (r=0.65, P<0.001) was observed between rate values of SR Ca²⁺ uptake and PThr¹⁷-PLB levels in diabetic WT and TGR. A similar correlation (r=0.72, P<0.001) was obtained when the respective values of Hoe 140-treated and untreated diabetic TGR were plotted. The PLB protein levels as well as the phosphorylation status of PLB at Ser¹⁶ of the LV did not differ significantly between the experimental groups.

View larger version (49K): [in this window] [in a new window]   Figure 2. Site-specific phosphorylation status of phospholamban (PLB) at amino acid residue Thr17 (PThr-PLB) in left ventricular homogenates of wild-type (WT) and hKLK1 transgenic rats (TGR) with severe STZ-induced diabetes mellitus. Quantification of the site-specific phosphorylation status of phospholamban (PLB) at amino acid residue Thr17 (PThr-PLB) of nondiabetic (A) and diabetic (B–D) wild-type (WT) and hKLK1 transgenic rats (TGR). C, D) Effect of treatment of diabetic transgenic rats with the bradykinin B2 receptor antagonist Hoe 140 on PThr17-PLB level. Each box plot relies on data of 6 to 8 different animals/group. *P < 0.05 vs. diabetic WT rats and Hoe140-treated diabetic TGR. Representative Western blots for monomeric PThr17-PLB (6.5 kDa signal) of 4 different animals in each group are shown below each box plot graph. Blots were probed with an antibody specifically directed against PThr17-PLB. Protein per lane: 10 µg.

CONCLUSIONS AND SIGNIFICANCE

Isolated diastolic dysfunction is an early manifestation of diabetic cardiomyopathy. Clinical and experimental evidence supports the concept that this is associated with abnormal cellular Ca²⁺ handling due to impairment of Ca²⁺ transport processes in cardiac membranes. The function of the SR Ca²⁺ pump SERCA2a is affected in particular. Kinins are of special interest: their potential NO-related free radical scavenging properties, endothelium-dependent vasodilating and antifibrotic actions, their stimulating effect on cardiac glucose uptake as well as their apparent capacity to increase SERCA2a expression observed under various pathological conditions may be beneficial in diabetic cardiopathy.

In this study, we used TGR with cardiac expression of hKLK to investigate the influence of a chronically activated tissue KLK system on contractile performance and SR Ca²⁺ handling in STZ-induced diabetic cardiomyopathy. Our findings indicate that 1) in diabetic cardiomyopathy, targeting the cardiac KLK expression was beneficial for defective systolic and diastolic performance; 2) diabetic TGR(hKLK1), in contrast to diabetic WT, showed no significant impairment of SR Ca²⁺ transport activity; 3) improved SR Ca²⁺ uptake in diabetic TGR(hKLK1) was linked to increased phosphorylation of PLB at the CaM kinase-specific residue Thr¹⁷; 4) these transgene-related beneficial effects were at least partly abolished by treatment with the BK B₂ receptor antagonist icatibant. The findings suggest that expression of a tissue KLK transgene can protect the rat heart at least in part against diabetes-specific contractile disturbances due to rescued SR Ca²⁺ handling. Thereby, a B₂ receptor-mediated process leading to enhanced phosphorylation of PLB at Thr¹⁷ is involved. The hypothetical schematic diagram in Fig. 3 postulates this is due to activation of a putative BK inositol 1,4,5-trisphosphate Ca²⁺ release CaM kinase signaling pathway in the myocardium of diabetic TGR expressing the BK-forming enzyme hKLK1 (Fig. 3) . The increase of Thr¹⁷-PLB phosphorylation observed in diabetic TGR(hKLK1) did not affect the apparent Ca²⁺ affinity of the Ca²⁺-transporting ATPase of the SR. This does not agree with the generally accepted view that CaM kinase-dependent PLB phosphorylation produces an increase in the Ca²⁺ affinity of the SR Ca²⁺ ATPase. Because the latter view is based on investigations in nondiabetic animals only, our finding in diabetic rats may indicate that this PLB phosphorylation alters the kinetic properties of the SR Ca²⁺-ATPase in the nondiabetic and diabetic myocardium in a different manner.

View larger version (30K): [in this window] [in a new window]   Figure 3. Hypothetical schematic diagram showing the possible cardiac signaling pathway responsible for accelerated SERCA2a-catalyzed sarcoplasmic reticulum (SR) Ca2+ transport in transgenic diabetic rats expressing the tissue human (h) isoform 1 of the bradykinin (BK) -forming enzyme kallikrein (KLK). It assumes an additional activation of B2 receptors (B2-R) at the surface of cardiomyocytes by surplus amounts of BK, which are generated in the KLK reaction that is catalyzed by the transgene encoded hKLK1. This results in increased formation of inositol 1,4,5-trisphosphate (IP3) in cardiomyocytes of diabetic transgenic rats and causes an increased, probably local, release of Ca2+ from IP3-sensitive part of the SR. The rise of "signaling Ca2+" concentration in the vicinity of the reticular Ca2+/calmodulin-dependent protein kinase II (CaMKII) activates this enzyme, which in turn phosphorylates phospholamban (PLB) at residue Thr17 (P). This covalent modification of PLB abolishes inhibition at least partially of the PLB-controlled SR Ca2+ pump, results in faster removal of "activator Ca2+" from the myoplasma into the IP3-insensitive part of the SR and, hence, accelerates impaired relaxation of the diabetic myocardium. Note: An increased expression of the otherwise low abundant IP3-operated Ca2+ release channels (IP3R) in the diabetic myocardium, which has not been shown so far, would be a prerequisite for the functional effective operation of this signaling pathway in the diabetic rather than the nondiabetic heart of transgene hKLK1 expressers.

In conclusion, the results of our study demonstrate that a potentiated kallikrein-kinin system is able to counteract at least partially the development of contractile dysfunction in STZ-induced diabetes, an improvement obviously linked to rescued SR Ca²⁺ transport.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1614fje;

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