OBJECTIVE

Diabetic cardiomyopathy is associated with increased mortality in patients with diabetes. The underlying pathology of this disease is still under discussion. We studied the role of the kinin B1 receptor on the development of experimental diabetic cardiomyopathy.

RESEARCH DESIGN AND METHODS

We utilized B1 receptor knockout mice and investigated cardiac inflammation, fibrosis, and oxidative stress after induction of streptozotocin (STZ)-induced diabetes. Furthermore, the left ventricular function was measured by pressure-volume loops after 8 weeks of diabetes.

RESULTS

B1 receptor knockout mice showed an attenuation of diabetic cardiomyopathy with improved systolic and diastolic function in comparison with diabetic control mice. This was associated with a decreased activation state of the mitogen-activated protein kinase p38, less oxidative stress, as well as normalized cardiac inflammation, shown by fewer invading cells and no increase in matrix metalloproteinase-9 as well as the chemokine CXCL-5. Furthermore, the profibrotic connective tissue growth factor was normalized, leading to a reduction in cardiac fibrosis despite severe hyperglycemia in mice lacking the B1 receptor.

CONCLUSIONS

These findings suggest that the B1 receptor is detrimental in diabetic cardiomyopathy in that it mediates inflammatory and fibrotic processes. These insights might have useful implications on future studies utilizing B1 receptor antagonists for treatment of human diabetic cardiomyopathy.

Diabetic cardiomyopathy, as it occurs in patients with diabetes, carries a substantial risk concerning the subsequent development of heart failure and increased mortality (1). Different pathophysiological stimuli are involved in its development and mediate tissue injury leading to left ventricular systolic and diastolic dysfunction. Accumulation of cardiac fibrosis with distinct changes in the regulation of the extracellular matrix (2,3), excessive generation of reactive oxygen species (4), and cardiac inflammation (5,6), characterized by increased levels of proinflammatory cytokines and transendothelial migration of immunocompetent cells, plays a role in the manifestation of diabetic cardiomyopathy. Experimental stimulation of the local tissue kallikrein-kinin system has been shown to be beneficial in different forms of cardiomyopathies (7,,,11). Most of these effects are attributed to the kinin B2 receptor (B2R), while the role of the kinin B1 receptor (B1R) in cardiac failure is still under discussion. In contrast to the B2R, which is constitutively expressed in the cardiac tissue, the B1R is expressed at very low levels under basal conditions. Nevertheless, it is highly inducible under pathological conditions by pathological mediators such as bacterial lipopolysaccharide (12), cytokines (13), and ischemia but also by hyperglycemia (14), as can be shown in different animal models of cardiomyopathy. Also, in endomyocardial biopsies of patients with end-stage heart failure, this upregulation could be demonstrated and correlated with increased expression of proinflammatory cytokines in those patients (15). Whether B1R upregulation is cardioprotective, parallel to that of the B2R (16,17), or is cardiotoxic (13,18,19) remains debated. To further clarify the role of the B1R in the pathogenesis of diabetic cardiomyopathy, we investigated the left ventricular function in an animal model of streptozotocin (STZ)-induced type 1 diabetes using B1R knockout mice. Furthermore, changes in the left ventricular remodeling, inflammation, and oxidative stress were analyzed.

Twenty-five B1R knockout mice (B1R−/−) on a C57/BL6 genetic background and 25 littermates (B1R+/+) aged 2 months were obtained from the Max-Delbrück Center for Molecular Medicine (Berlin-Buch, Germany) (13). Diabetes was induced by injection of STZ (50 mg/kg i.p. for 5 days) in 15 B1R−/−(B1R−/−-STZ) and 15 C57/BL6 mice (B1R+/+-STZ), while the others served as nondiabetic controls (B1R−/−and control). Hyperglycemia (glucose >22 mmol/l) was confirmed 7 days later using a reflectance meter (Acutrend; Boehringer, Mannheim, Germany), as well as at the end of the study (glucose >30 mmol/l). The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publ. no. 85-23, revised 1985).

Surgical procedures and hemodynamic measurements.

Eight weeks after induction of diabetes, left ventricular function was analyzed using pressure-volume loops. The animals were anesthetized (125 mg/g i.p. thiopental), intubated, and artificially ventilated. As described recently (20), a 1.2-F microconductance pressure catheter (SciSense, Ontario, Canada) was positioned in the left ventricle for registration of left ventricular pressure-volume loops in a closed-chest model. Indexes of cardiac function were derived from pressure-volume data obtained both at steady state and during transient preload reduction by occlusion. Systolic function was quantified by left ventricual end systolic pressure (mmHg), by dP/dtmax (mmHg/s), and by ejection fraction (%). Global cardiac function was quantified by the end systolic volume (μl), end diastolic volume (μl), stroke volume (μl), cardiac output (μl/min), the ratio of cardiac output to body weight (ml · min−1 · g−1), and heart rate (beats/min). Diastolic function was measured by left ventricular end diastolic pressure (mmHg), left ventricular pressure fall (dP/dtmin) (mmHg/s), and diastolic stiffness. Diastolic stiffness was calculated from the end diastolic pressure-volume relationship [EDP = C · exp (b − Ved)] where b is for stiffness (21). Moreover, mean arterial blood pressure was analyzed from measurements in the arteria carotis (22). Cardiac tissue was harvested and snap frozen for later measurements. All following measurements were performed in 10 animals per group.

Histological measurements.

Immunohistochemistry was carried out using primary antibodies for collagen type 1 and type 3, the connective tissue growth factor (CTGF), the matrix-metalloproteinase-9 (MMP-9), and CD3+, CD11b+, CD45+, and CD68+cells as well as nitrotyrosine and myeloperoxidase (MPO) (all from Serotec, Munich, Germany) followed by the DAKO Envision horseradish peroxidase technique (DAKO, Glostrup, Denmark). Histological costainings were performed using prinamry antibodies for CD68, tumor necrosis factor (TNF)-α (R&D Systems, Wiesbaden, Germany), sarcomeric actin, and TAB-1 (Cell Signaling Technology, Danvers, MA).

Real-time RT-PCR.

Real-time RT-PCR (ABI Prism 7900 HT Sequence Detection System software, version 2.2.2.; Perkin Elmer) was carried out as previously described (23) using primers for B1R and B2R, the cytokine interleukin (IL)-1β, TNF-α, and IL-6, as well as for the chemokine CXCL-5. 18S was used as a housekeeping gene.

Western blot for evaluation of total p38 mitogen-activated protein kinase and p38 phosphorylation.

Total p38 mitogen-activated protein kinase (MAPK) and phosphorylated p38 MAPK were detected with each specific antibody. Moreover, TAB-1 (all from Cell Signaling Technology) was detected. The blots were visualized with a chemiluminescene system (Amersham Bioscience, Buckinghamshire, U.K.). Quantitative analysis of the intensity of the bands was performed with NIH Image 1.63 software (National Institutes of Health, Bethesda, MD).

Statistical analysis.

All data are expressed as means ± SE. Statistical significance between multiple groups was determined using ANOVA and post hoc analysis with a Bonferroni test. Values of P < 0.05 were considered significant.

Eight weeks after induction of STZ-induced diabetes, glucose levels were found to be highly increased in B1R−/−-STZ and B1R+/+-STZ mice but did not differ between both diabetic groups. Body weight decreased in both groups when compared with controls (Table 1).

TABLE 1
B1R+/+B1R−/−B1R+/+-STZB1R−/−-STZ
Body weight (g) 27 ± 1 28 ± 1 17 ± 1* 17 ± 1* 
Glucose levels (mmol/l) 6 ± 0.2 6 ± 0.2 31 ± 2* 32 ± 2* 
Hemodynamic function     
    Heart rate (beats/min) 465 ± 22 486 ± 32 302 ± 27* 389 ± 35 
    End diastolic volume (μl) 53 ± 2 48 ± 4 49 ± 4 47 ± 3 
    End systolic volume (μl) 18 ± 4 15 ± 3 28 ± 5 23 ± 5 
    Stroke volume (μl) 35 ± 6 31 ± 4 19 ± 6* 24 ± 5 
    Cardiac output (ml/min) 16.1 ± 1 15.8 ± 1 6.3 ± 0.6* 9.3 ± 1 
    Cardiac output/body weight (ml · min−1· g−10.61 ± 0.02 0.57 ± 0.04 0.41 ± 0.04* 0.56 ± 0.03 
    Left ventricular systolic pressure (mmHg) 98 ± 4 103 ± 5 72 ± 5* 86 ± 6 
    dP/dtmax(mmHg/s) 6,658 ± 346 6,858 ± 256 3,215 ± 201* 5,214 ± 286 
    Ejection fraction (%) 66 ± 4 64 ± 5 38 ± 7* 51 ± 4 
    Left ventricular diastolic pressure (mmHg) 2.5 ± 1 2.8 ± 1 9.4 ± 2* 4.4 ± 2 
    dP/dtmin(mmHg/s) −5,896 ± 301 −5,485 ± 285 −2,248 ± 247* −4,257 ± 244* 
    Left ventricular stiffness (ml−10.027 ± 0.002 0.034 ± 0.002 0.127 ± 0.001* 0.047 ± 0.004* 
    Mean blood pressure (mmHg) 95 ± 4 98 ± 5 68 ± 7* 88 ± 6* 
B1R+/+B1R−/−B1R+/+-STZB1R−/−-STZ
Body weight (g) 27 ± 1 28 ± 1 17 ± 1* 17 ± 1* 
Glucose levels (mmol/l) 6 ± 0.2 6 ± 0.2 31 ± 2* 32 ± 2* 
Hemodynamic function     
    Heart rate (beats/min) 465 ± 22 486 ± 32 302 ± 27* 389 ± 35 
    End diastolic volume (μl) 53 ± 2 48 ± 4 49 ± 4 47 ± 3 
    End systolic volume (μl) 18 ± 4 15 ± 3 28 ± 5 23 ± 5 
    Stroke volume (μl) 35 ± 6 31 ± 4 19 ± 6* 24 ± 5 
    Cardiac output (ml/min) 16.1 ± 1 15.8 ± 1 6.3 ± 0.6* 9.3 ± 1 
    Cardiac output/body weight (ml · min−1· g−10.61 ± 0.02 0.57 ± 0.04 0.41 ± 0.04* 0.56 ± 0.03 
    Left ventricular systolic pressure (mmHg) 98 ± 4 103 ± 5 72 ± 5* 86 ± 6 
    dP/dtmax(mmHg/s) 6,658 ± 346 6,858 ± 256 3,215 ± 201* 5,214 ± 286 
    Ejection fraction (%) 66 ± 4 64 ± 5 38 ± 7* 51 ± 4 
    Left ventricular diastolic pressure (mmHg) 2.5 ± 1 2.8 ± 1 9.4 ± 2* 4.4 ± 2 
    dP/dtmin(mmHg/s) −5,896 ± 301 −5,485 ± 285 −2,248 ± 247* −4,257 ± 244* 
    Left ventricular stiffness (ml−10.027 ± 0.002 0.034 ± 0.002 0.127 ± 0.001* 0.047 ± 0.004* 
    Mean blood pressure (mmHg) 95 ± 4 98 ± 5 68 ± 7* 88 ± 6* 

Data are means ± SE. Hemodynamic function of control mice (B1R+/+) or mice lacking the B1R (B1R−/−) under basal conditions and 8 weeks after induction of STZ-induced diabetes.

*P< 0.05 vs. nondiabetic controls;

P< 0.05 vs. B1R+/+-STZ. dP/dtmax, contractility; dP/dtmin, relaxation.

Hemodynamic data.

Lack of B1R had no effect on cardiac function under normoglycemic conditions. The heart rate was significantly decreased in B1R+/+-STZ mice when compared with controls, due to the known effect of diabetic cardiac autonomy (24), while B1R−/−-STZ mice were not statistically different from their controls. No ventricular dilatation was demonstrated in either STZ group when compared with their controls, while stroke volume was smaller in STZ, which contributed to impaired cardiac output in comparison with the controls. This decline of cardiac output could be partly prevented by B1R knockout but was still impaired when B1R−/−-STZ mice were compared with controls. The systolic and diastolic parameters end systolic pressure, dP/dtmax, and ejection fraction were significantly decreased; the end diastolic pressure as well as diastolic stiffness was increased when STZ was compared with the control group. However, the impairment in these parameters was much less pronounced in B1R−/−-STZ mice (Table 1 and Fig. 1).

FIG. 1.

Representative pressure-volume loops during a preload reduction of control mice (B1R+/+) or mice lacking the B1R (B1R−/−) under basal conditions and 8 weeks after induction of STZ-induced diabetes. The thick black line at the bottom indicates the left ventricular stiffness, which is increased in B1R+/+-STZ (indicated by the thick black arrow).

FIG. 1.

Representative pressure-volume loops during a preload reduction of control mice (B1R+/+) or mice lacking the B1R (B1R−/−) under basal conditions and 8 weeks after induction of STZ-induced diabetes. The thick black line at the bottom indicates the left ventricular stiffness, which is increased in B1R+/+-STZ (indicated by the thick black arrow).

Close modal

Cardiac inflammation and oxidative stress.

In the myocardium of the diabetic mice, the mRNA abundance of the proinflammatory cytokines IL-1β, IL-6, and TNF-α as well as the chemokine CXCL-5 were significantly increased by B1R+/+-STZ compared with controls (Fig. 2). This was associated with increased numbers of CD3+, CD11b+, CD45+, and CD68+cells (Fig. 3) and the protein abundance of MMP-9 (Fig. 4). This upregulation was prevented by B1R−/−-STZ, resulting in normalized levels compared with controls. Moreover, a major part of TNF-α is produced by inflammatory cells (CD68) (Fig. 5). The abundance of nitrotyrosine and myeloperoxidase was increased in the cardiac tissue of the STZ group, as an indicator of increased oxidative stress (Fig. 4). The lack of the B1R reduced this increased expression of nitrotyrosine and myeloperoxidase in comparison with STZ, despite severe hyperglycemia when B1R−/−-STZ was compared with B1R+/+-STZ (Fig. 4).

FIG. 2.

mRNA levels of cardiac cytokines in control mice (B1R+/+) or mice lacking the B1R (B1R−/−) under basal conditions and 8 weeks after induction of STZ-induced diabetes with increased levels of IL-1β, IL-6, and TNF-α in STZ measured by real-time RT-PCR. Moreover, the figure shows mRNA levels of the chemokine CXCL-5 levels as well as mRNA levels of the B1R and B2R. *P < 0.05 vs. B1R+/+ and B1R−/− STZ. #P < 0.05 vs. B1R+/+.

FIG. 2.

mRNA levels of cardiac cytokines in control mice (B1R+/+) or mice lacking the B1R (B1R−/−) under basal conditions and 8 weeks after induction of STZ-induced diabetes with increased levels of IL-1β, IL-6, and TNF-α in STZ measured by real-time RT-PCR. Moreover, the figure shows mRNA levels of the chemokine CXCL-5 levels as well as mRNA levels of the B1R and B2R. *P < 0.05 vs. B1R+/+ and B1R−/− STZ. #P < 0.05 vs. B1R+/+.

Close modal
FIG. 3.

Increased inflammatory cells (CD3+, CD11+, CD45+, and CD68+) in the cardiac tissue of B1R+/+-STZ with representative pictures of all groups for CD11+ and CD68+ cells in control mice (B1R+/+) or mice lacking the B1R (B1R−/−) under basal conditions and 8 weeks after induction of STZ-induced diabetes. *P < 0.05 vs. B1R+/+ and B1R−/− STZ. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Increased inflammatory cells (CD3+, CD11+, CD45+, and CD68+) in the cardiac tissue of B1R+/+-STZ with representative pictures of all groups for CD11+ and CD68+ cells in control mice (B1R+/+) or mice lacking the B1R (B1R−/−) under basal conditions and 8 weeks after induction of STZ-induced diabetes. *P < 0.05 vs. B1R+/+ and B1R−/− STZ. (A high-quality digital representation of this figure is available in the online issue.)

Close modal
FIG. 4.

Increased protein levels of oxidative stress (nitrotyrosin and MPO) as well as the MMP-9 in the cardiac tissue of B1R+/+-STZ. Moreover, the figure shows protein levels and mRNA levels of eNOS and representative pictures of all groups for MPO and eNOS. *P < 0.05 vs. B1R+/+. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Increased protein levels of oxidative stress (nitrotyrosin and MPO) as well as the MMP-9 in the cardiac tissue of B1R+/+-STZ. Moreover, the figure shows protein levels and mRNA levels of eNOS and representative pictures of all groups for MPO and eNOS. *P < 0.05 vs. B1R+/+. (A high-quality digital representation of this figure is available in the online issue.)

Close modal
FIG. 5.

Immunofluorescent stainings of cardiac tissue of a diabetic wild-type mouse with stainings for CD68, TNF-α, as well α-sacromeric actin and DAPI (for cell nuclei) showing that TNF-α is secreted by inflammatory CD68+ cells. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Immunofluorescent stainings of cardiac tissue of a diabetic wild-type mouse with stainings for CD68, TNF-α, as well α-sacromeric actin and DAPI (for cell nuclei) showing that TNF-α is secreted by inflammatory CD68+ cells. (A high-quality digital representation of this figure is available in the online issue.)

Close modal

Protein levels of endothelial nitric oxide synthase levels (eNOS) were downregulated in both diabetic groups. Nevertheless, the mRNA content was only downregulated significantly in the the B1R+/+-STZ but not in the B1R−/−-STZ mice (Fig. 4). Furthermore, the phosphorylation state of the MAPK p38, known to contribute to tissue inflammation, was increased in STZ mice compared with controls, again an effect that was reduced in B1R−/−-STZ mice when compared with controls. Moreover, TAB-1 protein was significantly inreased in diabetic wild-type compared with B1R−/−-STZ mice (Fig. 6).

FIG. 6.

A: Immunofluorescent stainings of cardiac tissue of B1R+/+-STZ and B1R−/−-STZ showing protein levels of TAK-1 binding protein (TAB-1) and α-sacromeric actin as well as DAPI (for cell nuclei). This demonstrates a reduced protein content of TAB-1 in B1R−/−-STZ. B: Quantification of protein levels of the MAPK p38 and its phosphorylated form as well as TAB-1 showing a normalization of the p38 activation and the TAB-1 protein content in B1R−/−-STZ compared with B1R+/+-STZ. *P < 0.05 vs. B1R−/−-STZ. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

A: Immunofluorescent stainings of cardiac tissue of B1R+/+-STZ and B1R−/−-STZ showing protein levels of TAK-1 binding protein (TAB-1) and α-sacromeric actin as well as DAPI (for cell nuclei). This demonstrates a reduced protein content of TAB-1 in B1R−/−-STZ. B: Quantification of protein levels of the MAPK p38 and its phosphorylated form as well as TAB-1 showing a normalization of the p38 activation and the TAB-1 protein content in B1R−/−-STZ compared with B1R+/+-STZ. *P < 0.05 vs. B1R−/−-STZ. (A high-quality digital representation of this figure is available in the online issue.)

Close modal

Kinin receptor regulation.

The B1R mRNA was increased by STZ-induced diabetes in wild-type mice. The B2R mRNA was also increased due to diabetic condition in wild-type mice. In B1R−/−animals, the B2R expression was higher compared with wild-type mice under basal conditions. Interestingly, there was no further B2R mRNA upregulation due to diabetic conditions as seen in wild-type mice (Fig. 2).

Cardiac fibrosis.

CTGF was highly increased in diabetic animals. This increase in CTGF was accompanied by increased levels of collagen type 1 and 3, as an indicator of cardiac fibrosis. In contrast, CTGF was normalized in B1R−/−-STZ, which translated into normalized collagen type 1 and type 3 levels as well, when compared with the controls (Fig. 7).

FIG. 7.

Increased levels of CTGF (A) and collagen type 1 (B) and 3 (C) in cardiac tissue of B1R+/+-STZ compared with nondiabetic controls. B1R−/−-STZ have normalized cardiac fibrosis as well as normalized levels of CTGF. *P < 0.05 vs. B1R+/+- and B1R−/−-STZ.

FIG. 7.

Increased levels of CTGF (A) and collagen type 1 (B) and 3 (C) in cardiac tissue of B1R+/+-STZ compared with nondiabetic controls. B1R−/−-STZ have normalized cardiac fibrosis as well as normalized levels of CTGF. *P < 0.05 vs. B1R+/+- and B1R−/−-STZ.

Close modal

The salient finding of this study is that B1R gene deletion attenuates cardiac systolic and diastolic dysfunction in experimental diabetic cardiomyopathy. Diabetic cardiomyopathy is characterized by an increase in the phosphorylation state of the MAPK p38, which was associated with augmented cardiac inflammation, cardiac fibrosis, and oxidative stress in cardiac tissue. These changes were normalized in mice lacking the B1R, despite the occurrence of comparable severe hyperglycemia.

Experimental stimulation of the kallikrein-kinin system by gene transfer (25,26) and/or by the use of transgenic kallikrein overexpressing animals (10) attenuates diabetic cardiomyopathy. This is in agreement with other studies showing potent cardioprotective effects of the kallikrein-kinin system in animal models of ischemic (27,29), pressure overload (8), and septic (12) and hypertensive (9) cardiomyopathy. We, and others, using the STZ model of diabetes, were able to show that these cardiobeneficial effects of kallikrein-kinin mediate anti-inflammatory and antifibrotic effects and, furthermore, reduce oxidative stress (10,30,31) as well as improve glucose utilization and lipid metabolism (26,32). Both receptors of the system, the B1R and the B2R, are upregulated in the diabetic heart (14). The cardioprotective effects are mediated mainly by the B2R, since pharmacological inhibition of the B2R was seen to abolish these cardioprotective effects (31). The relationship between B1R and the development of heart failure is still under investigation. Recently, it was shown that the B1R may yield similar effects when compared with the B2R in an animal model of myocardial infarction (17). Nonetheless, other researchers have shown that a lack of the B1R reduced infarct size in ischemia reperfusion injury (19,33), a finding that indeed may imply an opposite function compared with that of the B2R. While there is good evidence that the B1R plays a detrimental role in autonomic diabetic nociception (34), obstructive nephropathy (35), and stroke (36) by modulating inflammatory processes and increasing inflammation, its role in the development of diabetic cardiomyopathy has not yet been directly investigated.

We demonstrated recently that diabetic cardiomyopathy is associated with increased cardiac inflammation (2,5,10). These inflammatory processes were associated with increased oxidative stress and cardiac fibrosis, all contributing to systolic and diastolic dysfunction under diabetic conditions. Since the B1R is known to be upregulated by IL-1β (37) and hyperglycemia (14,38) and mediate tissue inflammation by increasing invading cells and proinflammatory cytokines in airway diseases (39), we investigated its role during the development of diabetic cardiomyopathy. On one hand, in nondiabetic mice with gene deletion of the B1R, the cardiac function remained unchanged when this was compared with control mice in the current study. That is a finding that is in agreement with others (17,40,42). On the other hand, attenuated cardiac dysfunction despite severe hyperglycemia indicates a detrimental role of the B1R in diabetic cardiomyopathy when systolic and diastolic function of B1R−/−-STZ were compared with STZ.

One intracellular pathway of the B1R was shown to be dependent on the MAPK p38 (43). The current study shows an activation of the p38 pathway in the STZ group, which was normalized in the B1R−/−-STZ group. P38 phosphorylation can be induced by TAK-1 binding protein (44). In line with these findings, increased protein content of TAB-1 in diabetic wild-type mice compared with B1R−/−-STZ mice could be documented here. Since p38 activation plays a role in diabetic cardiomyopathy by inducing cardiac inflammation (45), we documented increased cardiac levels of cytokines in the STZ group, namely of Il-1β, IL-6, and TNF-α, known to cause myocardial dysfunction and mediate leukocyte infiltration during tissue inflammation (46). This cytokine induction was decreased in diabetic mice lacking the B1R.

Moreover, the number of invading immunocompetent cells was increased in the STZ group, thus yielding another marker of cardiac inflammation. These increased numbers were normalized in mice lacking the B1R. This is important, since those invading cells are one major source of cytokine production within the cardiac tissue (e.g., as shown here by the colocalization of TNF-α with CD68+cells). Furthermore, we show that the protein levels of MMP-9 were also increased in STZ and decreased in diabetic mice with gene deletion of the B1R. Recent findings (47) suggest that especially MMP-9 modulates the transendothelial migration of leukocytes from the vessel to the tissue, where an inflammatory progress is ongoing. This can be explained by the fact that MMP-9 does not solely cleave gelatin but indeed processes and activates many chemokines and cytokines and thereby directly modulates inflammation. In line with these effects, it was shown in a mouse model of hepatitis that MMP-9 knockout mice were protected against invading leukocytes undergoing transendothelial migration (48). Only recently could it be shown that induction of MMP-9 expression is triggered by bradykinin, using cell culture rat astrocytes (49). Although those authors showed that B2R antagonism inhibited this increase (49), our data also suggest that the B1R plays a role in MMP-9 expression in cardiac tissue under diabetic conditions. Moreover, recently, the B1R was shown to be essential for IL-1β–driven cell recruitment of immunocompetent cells by inducing the chemokine CXCL-5 in endothelial cells (50). This recruitment of CXCL-5, known to be one important player in leukocyte recruitment to sites of tissue inflammation, was abolished in mice without the B1R or when a pharmacological B1R antagonist was applied (50). In line with these data, we show here that the chemokine CXCL-5 was increased under diabetic conditions. This effect was completely normalized in mice lacking the B1R despite severe hyperglycemia. The normalized levels of CXCL-5 and MMP-9 result in reduced migration of inflammatory cells into the cardiac tissue. Therefore, inflammation leading to cardiac damage due to invasion of these cells was attenuated in diabetic B1R−/−-STZ mice compared with diabetic controls with increased levels of CXCL-5 and MMP-9.

Furthermore, it was shown that the B1R, but not the B2R, increases the mRNA abundance of the profibrotic CTGF and thereby increases collagen mRNA and protein production in human fibroblasts, an effect that could be blocked by a B1R antagonist (51). Following these findings, we show increased levels of CTGF in the cardiac tissue of STZ mice, leading to increased collagen accumulation, which is known to be a hallmark in the development of diabetic cardiomyopathy leading to increased cardiac stiffness contributing to diastolic and systolic failure. Together with those findings, this mechanism could not be observed in mice with B1R gene deletion, thus showing no increase in CTGF or collagen accumulation.

Much evidence has indicated that oxidative stress plays an important role in the failing diabetic heart (52). This can be attenuated by the kallikrein-kinin system (8,10). Consistently, we show increased nitrotyrosine and myeloperoxidase protein levels (expressed from inflammatory cells) in the cardiac tissue of the STZ group. Together with reduced inflammatory cells, nitrotyrosine and myeloperoxidase were reduced in the B1R−/−-STZ group. These data suggest that the B1R might play a role in the generation of oxidative stress, most probably due to the increased recruitment of inflammatory cells resulting in increased myeloperoxidase present in the cardiac tissue. Future studies have to investigate whether this effect is only mediated by the B1R or if changes in basal B2R level expression, as shown in this study, are also influencing generation of oxidative stress in the B1R−/−-STZ group.

The protein content of eNOS, known to exert antioxidative effects, was similarly reduced in both diabetic groups. Interestingly, B1R−/−-STZ showed no significant downregulation on mRNA levels. Despite these changes in mRNA content, which may be explained by posttranscriptional modification, these data suggest that the diabetes-induced downregulation of eNOS protein is not mainly regulated by the B1R knockout. Again, further studies have to reveal the impact of B2R regulation on these effects. In conclusion, this study demonstrates that a lack of the B1R attenuates the development of STZ-induced diabetic cardiomyopathy with a decrease of cardiac inflammation, fibrosis, and oxidative stress.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This study was supported by the Deutsche Forschungsgemeinschaft (SFB-TR-19; A2, Z3).

No potential conflicts of interest relevant to this article were reported.

The authors thank M. Kastner for proofreading the final version of the manuscript.

1.
Poornima
IG
,
Parikh
P
,
Shannon
RP
:
Diabetic cardiomyopathy: the search for a unifying hypothesis
.
Circ Res
2006
; 
98
:
596
605
2.
Westermann
D
,
Rutschow
S
,
Jager
S
,
Linderer
A
,
Anker
S
,
Riad
A
,
Unger
T
,
Schultheiss
HP
,
Pauschinger
M
,
Tschope
C
:
Contributions of inflammation and cardiac matrix metalloproteinase activity to cardiac failure in diabetic cardiomyopathy: the role of angiotensin type 1 receptor antagonism
.
Diabetes
2007
; 
56
:
641
646
3.
Zhang
L
,
Cannell
MB
,
Phillips
AR
,
Cooper
GJ
,
Ward
ML
:
Altered calcium homeostasis does not explain the contractile deficit of diabetic cardiomyopathy
.
Diabetes
2008
; 
57
:
2158
2166
4.
Dorenkamp
M
,
Riad
A
,
Stiehl
S
,
Spillmann
F
,
Westermann
D
,
Du
J
,
Pauschinger
M
,
Noutsias
M
,
Adams
V
,
Schultheiss
HP
,
Tschope
C
:
Protection against oxidative stress in diabetic rats: role of angiotensin AT(1) receptor and beta 1-adrenoceptor antagonism
.
Eur J Pharmacol
2005
; 
520
:
179
187
5.
Li
J
,
Leschka
S
,
Rutschow
S
,
Schwimmbeck
PL
,
Husmann
L
,
Noutsias
M
,
Westermann
D
,
Poller
W
,
Zeichhardt
H
,
Klingel
K
,
Tschope
C
,
Schultheiss
HP
,
Pauschinger
M
:
Immunomodulation by interleukin-4 suppresses matrix metalloproteinases and improves cardiac function in murine myocarditis
.
Eur J Pharmacol
2007
; 
554
:
60
68
6.
Westermann
D
,
Van Linthout
S
,
Dhayat
S
,
Dhayat
N
,
Schmidt
A
,
Noutsias
M
,
Song
XY
,
Spillmann
F
,
Riad
A
,
Schultheiss
HP
,
Tschope
C
:
Tumor necrosis factor-alpha antagonism protects from myocardial inflammation and fibrosis in experimental diabetic cardiomyopathy
.
Basic Res Cardiol
2007
; 
102
:
500
507
7.
Yoshida
H
,
Zhang
JJ
,
Chao
L
,
Chao
J
:
Kallikrein gene delivery attenuates myocardial infarction and apoptosis after myocardial ischemia and reperfusion
.
Hypertension
2000
; 
35
:
25
31
8.
Li
HJ
,
Yin
H
,
Yao
YY
,
Shen
B
,
Bader
M
,
Chao
L
,
Chao
J
:
Tissue kallikrein protects against pressure overload-induced cardiac hypertrophy through kinin B2 receptor and glycogen synthase kinase-3beta activation
.
Cardiovasc Res
2007
; 
73
:
130
142
9.
Bledsoe
G
,
Chao
L
,
Chao
J
:
Kallikrein gene delivery attenuates cardiac remodeling and promotes neovascularization in spontaneously hypertensive rats
.
Am J Physiol Heart Circ Physiol
2003
; 
285
:
H1479
H1488
10.
Tschope
C
,
Walther
T
,
Escher
F
,
Spillmann
F
,
Du
J
,
Altmann
C
,
Schimke
I
,
Bader
M
,
Sanchez-Ferrer
CF
,
Schultheiss
HP
,
Noutsias
M
:
Transgenic activation of the kallikrein-kinin system inhibits intramyocardial inflammation, endothelial dysfunction, and oxidative stress in experimental diabetic cardiomyopathy
.
FASEB J
19
:
2057
2059
,
2005
11.
Chao
J
,
Yin
H
,
Gao
L
,
Hagiwara
M
,
Shen
B
,
Yang
ZR
,
Chao
L
:
Tissue kallikrein elicits cardioprotection by direct kinin b2 receptor activation independent of kinin formation
.
Hypertension
2008
; 
52
:
715
720
12.
Cayla
C
,
Todiras
M
,
Iliescu
R
,
Saul
VV
,
Gross
V
,
Pilz
B
,
Chai
G
,
Merino
VF
,
Pesquero
JB
,
Baltatu
OC
,
Bader
M
:
Mice deficient for both kinin receptors are normotensive and protected from endotoxin-induced hypotension
.
FASEB J
2007
; 
21
:
1689
1698
13.
Pesquero
JB
,
Araujo
RC
,
Heppenstall
PA
,
Stucky
CL
,
Silva
JA
 Jr
,
Walther
T
,
Oliveira
SM
,
Pesquero
JL
,
Paiva
AC
,
Calixto
JB
,
Lewin
GR
,
Bader
M
:
Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors
.
Proc Natl Acad Sci U S A
2000
; 
97
:
8140
8145
14.
Spillmann
F
,
Altmann
C
,
Scheeler
M
,
Barbosa
M
,
Westermann
D
,
Schultheiss
HP
,
Walther
T
,
Tschope
C
:
Regulation of cardiac bradykinin B1- and B2-receptor mRNA in experimental ischemic, diabetic, and pressure-overload-induced cardiomyopathy
.
Int Immunopharmacol
2002
; 
2
:
1823
1832
15.
Liesmaa
I
,
Kuoppala
A
,
Shiota
N
,
Kokkonen
JO
,
Kostner
K
,
Mayranpaa
M
,
Kovanen
PT
,
Lindstedt
KA
:
Increased expression of bradykinin type-1 receptors in endothelium of intramyocardial coronary vessels in human failing hearts
.
Am J Physiol Heart Circ Physiol
2005
; 
288
:
H2317
H2322
16.
Emanueli
C
,
Bonaria Salis
M
,
Stacca
T
,
Pintus
G
,
Kirchmair
R
,
Isner
JM
,
Pinna
A
,
Gaspa
L
,
Regoli
D
,
Cayla
C
,
Pesquero
JB
,
Bader
M
,
Madeddu
P
:
Targeting kinin B(1) receptor for therapeutic neovascularization
.
Circulation
2002
; 
105
:
360
366
17.
Xu
J
,
Carretero
OA
,
Sun
Y
,
Shesely
EG
,
Rhaleb
NE
,
Liu
YH
,
Liao
TD
,
Yang
JJ
,
Bader
M
,
Yang
XP
:
Role of the B1 kinin receptor in the regulation of cardiac function and remodeling after myocardial infarction
.
Hypertension
2005
; 
45
:
747
753
18.
Ni
A
,
Yin
H
,
Agata
J
,
Yang
Z
,
Chao
L
,
Chao
J
:
Overexpression of kinin B1 receptors induces hypertensive response to des-Arg9-bradykinin and susceptibility to inflammation
.
J Biol Chem
2003
; 
278
:
219
225
19.
Yin
H
,
Chao
J
,
Bader
M
,
Chao
L
:
Differential role of kinin B1 and B2 receptors in ischemia-induced apoptosis and ventricular remodeling
.
Peptides
2007
; 
28
:
1383
1389
20.
Westermann
D
,
Knollmann
BC
,
Steendijk
P
,
Rutschow
S
,
Riad
A
,
Pauschinger
M
,
Potter
JD
,
Schultheiss
HP
,
Tschope
C
:
Diltiazem treatment prevents diastolic heart failure in mice with familial hypertrophic cardiomyopathy
.
Eur J Heart Fail
2006
; 
8
:
115
121
21.
Westermann
D
,
Kasner
M
,
Steendijk
P
,
Spillmann
F
,
Riad
A
,
Weitmann
K
,
Hoffmann
W
,
Poller
W
,
Pauschinger
M
,
Schultheiss
HP
,
Tschope
C
:
Role of left ventricular stiffness in heart failure with normal ejection fraction
.
Circulation
2008
; 
117
:
2051
2060
22.
Westermann
D
,
Riad
A
,
Lettau
O
,
Roks
A
,
Savvatis
K
,
Becher
PM
,
Escher
F
,
Jan Danser
AH
,
Schultheiss
HP
,
Tschope
C
:
Renin inhibition improves cardiac function and remodeling after myocardial infarction independent of blood pressure
.
Hypertension
2008
; 
52
:
1068
1075
23.
Westermann
D
,
Van Linthout
S
,
Dhayat
S
,
Dhayat
N
,
Escher
F
,
Bucker-Gartner
C
,
Spillmann
F
,
Noutsias
M
,
Riad
A
,
Schultheiss
HP
,
Tschope
C
:
Cardioprotective and anti-inflammatory effects of interleukin converting enzyme inhibition in experimental diabetic cardiomyopathy
.
Diabetes
2007
; 
56
:
1834
1841
24.
Howarth
FC
,
Jacobson
M
,
Shafiullah
M
,
Adeghate
E
:
Effects of insulin treatment on heart rhythm, body temperature and physical activity in streptozotocin-induced diabetic rat
.
Clin Exp Pharmacol Physiol
2006
; 
33
:
327
331
25.
Emanueli
C
,
Salis
MB
,
Pinna
A
,
Stacca
T
,
Milia
AF
,
Spano
A
,
Chao
J
,
Chao
L
,
Sciola
L
,
Madeddu
P
:
Prevention of diabetes-induced microangiopathy by human tissue kallikrein gene transfer
.
Circulation
2002
; 
106
:
993
999
26.
Montanari
D
,
Yin
H
,
Dobrzynski
E
,
Agata
J
,
Yoshida
H
,
Chao
J
,
Chao
L
:
Kallikrein gene delivery improves serum glucose and lipid profiles and cardiac function in streptozotocin-induced diabetic rats
.
Diabetes
2005
; 
54
:
1573
1580
27.
Yao
YY
,
Yin
H
,
Shen
B
,
Chao
L
,
Chao
J
:
Tissue kallikrein infusion prevents cardiomyocyte apoptosis, inflammation and ventricular remodeling after myocardial infarction
.
Regul Pept
2007
; 
140
:
12
20
28.
Koch
M
,
Spillmann
F
,
Dendorfer
A
,
Westermann
D
,
Altmann
C
,
Sahabi
M
,
Linthout
SV
,
Bader
M
,
Walther
T
,
Schultheiss
HP
,
Tschope
C
:
Cardiac function and remodeling is attenuated in transgenic rats expressing the human kallikrein-1 gene after myocardial infarction
.
Eur J Pharmacol
2006
; 
550
:
143
148
29.
Agata
J
,
Chao
L
,
Chao
J
:
Kallikrein gene delivery improves cardiac reserve and attenuates remodeling after myocardial infarction
.
Hypertension
2002
; 
40
:
653
659
30.
Tschope
C
,
Spillmann
F
,
Rehfeld
U
,
Koch
M
,
Westermann
D
,
Altmann
C
,
Dendorfer
A
,
Walther
T
,
Bader
M
,
Paul
M
,
Schultheiss
HP
,
Vetter
R
:
Improvement of defective sarcoplasmic reticulum Ca2+ transport in diabetic heart of transgenic rats expressing the human kallikrein-1 gene
.
FASEB J
2004
; 
18
:
1967
1969
31.
Tschope
C
,
Walther
T
,
Koniger
J
,
Spillmann
F
,
Westermann
D
,
Escher
F
,
Pauschinger
M
,
Pesquero
JB
,
Bader
M
,
Schultheiss
HP
,
Noutsias
M
:
Prevention of cardiac fibrosis and left ventricular dysfunction in diabetic cardiomyopathy in rats by transgenic expression of the human tissue kallikrein gene
.
FASEB J
2004
; 
18
:
828
835
32.
Sharma
JN
,
Kesavarao
U
:
Cardiac kallikrein in hypertensive and normotensive rats with and without diabetes
.
Immunopharmacology
1996
; 
33
:
341
343
33.
Lagneux
C
,
Bader
M
,
Pesquero
JB
,
Demenge
P
,
Ribuot
C
:
Detrimental implication of B1 receptors in myocardial ischemia: evidence from pharmacological blockade and gene knockout mice
.
Int Immunopharmacol
2002
; 
2
:
815
822
34.
Gabra
BH
,
Berthiaume
N
,
Sirois
P
,
Nantel
F
,
Battistini
B
:
The kinin system mediates hyperalgesia through the inducible bradykinin B1 receptor subtype: evidence in various experimental animal models of type 1 and type 2 diabetic neuropathy
.
Biol Chem
2006
; 
387
:
127
143
35.
Klein
J
,
Gonzalez
J
,
Duchene
J
,
Esposito
L
,
Pradere
JP
,
Neau
E
,
Delage
C
,
Calise
D
,
Ahluwalia
A
,
Carayon
P
,
Pesquero
JB
,
Bader
M
,
Schanstra
JP
,
Bascands
JL
:
Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy
.
FASEB J
2009
; 
23
:
134
142
36.
Austinat
M
,
Braeuninger
S
,
Pesquero
JB
,
Brede
M
,
Bader
M
,
Stoll
G
,
Renne
T
,
Kleinschnitz
C
:
Blockade of bradykinin receptor B1 but not bradykinin receptor B2 provides protection from cerebral infarction and brain edema
.
Stroke
2009
; 
40
:
285
293
37.
Zhang
Y
,
Adner
M
,
Cardell
LO
:
IL-1beta-induced transcriptional up-regulation of bradykinin B1 and B2 receptors in murine airways
.
Am J Respir Cell Mol Biol
2007
; 
36
:
697
705
38.
Rodriguez
AI
,
Pereira-Flores
K
,
Hernandez-Salinas
R
,
Boric
MP
,
Velarde
V
:
High glucose increases B1-kinin receptor expression and signaling in endothelial cells
.
Biochem Biophys Res Commun
2006
; 
345
:
652
659
39.
Gama Landgraf
R
,
Jancar
S
,
Steil
AA
,
Sirois
P
:
Modulation of allergic and immune complex-induced lung inflammation by bradykinin receptor antagonists
.
Inflamm Res
2004
; 
53
:
78
83
40.
Duka
A
,
Kintsurashvili
E
,
Duka
I
,
Ona
D
,
Hopkins
TA
,
Bader
M
,
Gavras
I
,
Gavras
H
:
Angiotensin-converting enzyme inhibition after experimental myocardial infarct: role of the kinin B1 and B2 receptors
.
Hypertension
2008
; 
51
:
1352
1357
41.
Westermann
D
,
Lettau
O
,
Sobirey
M
,
Riad
A
,
Bader
M
,
Schultheiss
HP
,
Tschope
C
:
Doxorubicin cardiomyopathy-induced inflammation and apoptosis are attenuated by gene deletion of the kinin B1 receptor
.
Biol Chem
2008
; 
389
:
713
718
42.
Xu
J
,
Carretero
OA
,
Shesely
EG
,
Rhaleb
NE
,
Yang
JJ
,
Bader
M
,
Yang
XP
:
The kinin B1 receptor contributes to the cardioprotective effect of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in mice
.
Exp Physiol
2008
; 
94
:
322
329
43.
Ganju
P
,
Davis
A
,
Patel
S
,
Nunez
X
,
Fox
A
:
p38 stress-activated protein kinase inhibitor reverses bradykinin B(1) receptor-mediated component of inflammatory hyperalgesia
.
Eur J Pharmacol
2001
; 
421
:
191
199
44.
Lu
G
,
Kang
YJ
,
Han
J
,
Herschman
HR
,
Stefani
E
,
Wang
Y
:
TAB-1 modulates intracellular localization of p38 MAP kinase and downstream signaling
.
J Biol Chem
2006
; 
281
:
6087
6095
45.
Westermann
D
,
Rutschow
S
,
Van Linthout
S
,
Linderer
A
,
Bucker-Gartner
C
,
Sobirey
M
,
Riad
A
,
Pauschinger
M
,
Schultheiss
HP
,
Tschope
C
:
Inhibition of p38 mitogen-activated protein kinase attenuates left ventricular dysfunction by mediating pro-inflammatory cardiac cytokine levels in a mouse model of diabetes mellitus
.
Diabetologia
2006
; 
49
:
2507
2513
46.
Kristiansen
OP
,
Mandrup-Poulsen
T
:
Interleukin-6 and diabetes: the good, the bad, or the indifferent?
Diabetes
2004
; 
54
(
Suppl. 2
):
S114
S124
47.
Hu
J
,
Van den Steen
PE
,
Sang
QX
,
Opdenakker
G
:
Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases
.
Nat Rev Drug Discov
2007
; 
6
:
480
498
48.
Wielockx
B
,
Lannoy
K
,
Shapiro
SD
,
Itoh
T
,
Itohara
S
,
Vandekerckhove
J
,
Libert
C
:
Inhibition of matrix metalloproteinases blocks lethal hepatitis and apoptosis induced by tumor necrosis factor and allows safe antitumor therapy
.
Nat Med
2001
; 
7
:
1202
1208
49.
Hsieh
HL
,
Yen
MH
,
Jou
MJ
,
Yang
CM
:
Intracellular signalings underlying bradykinin-induced matrix metalloproteinase-9 expression in rat brain astrocyte-1
.
Cell Signal
2004
; 
16
:
1163
1176
50.
Duchene
J
,
Lecomte
F
,
Ahmed
S
,
Cayla
C
,
Pesquero
J
,
Bader
M
,
Perretti
M
,
Ahluwalia
A
:
A novel inflammatory pathway involved in leukocyte recruitment: role for the kinin B1 receptor and the chemokine CXCL5
.
J Immunol
2007
; 
179
:
4849
4856
51.
Ricupero
DA
,
Romero
JR
,
Rishikof
DC
,
Goldstein
RH
:
Des-Arg(10)-kallidin engagement of the B1 receptor stimulates type I collagen synthesis via stabilization of connective tissue growth factor mRNA
.
J Biol Chem
2000
; 
275
:
12475
12480
52.
Wang
J
,
Song
Y
,
Elsherif
L
,
Song
Z
,
Zhou
G
,
Prabhu
SD
,
Saari
JT
,
Cai
L
:
Cardiac metallothionein induction plays the major role in the prevention of diabetic cardiomyopathy by zinc supplementation
.
Circulation
2006
; 
113
:
544
554