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Increased kallikrein expression protects against cardiac ischemia¹

YIGAL M. PINTO², MICHAEL BADER^*, JOÃO B. PESQUERO^*,, CARSTEN TSCHÖPE, EGBERT SCHOLTENS, WIEK H. VAN GILST and HENDRIK BUIKEMA

Departments of Cardiology and Clinical Pharmacology, University Hospital Groningen, Groningen, The Netherlands;
^* Max-Delbrück Center for Molecular Medicine, Berlin-Buch, and
Department of Cardiology, University Hospital Benjamin Franklin, Free University, Berlin, Germany; and
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil

²Correspondence: University Hospital Groningen, Department of Cardiology, Secretary: Gretha Beuker, zone S4 kamer 4.252, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. E-mail: y.m.pinto{at}thorax.azg.nl

SPECIFIC AIM

We set out to evaluate whether a primary increase of bradykinin in the absence of angiotensin-converting enzyme (ACE) inhibition is sufficient to protect against cardiac ischemia.

Kinins have been suggested to protect against cardiac ischemia, but direct proof for this idea is still lacking. The idea that kinins can protect against cardiac ischemia stems from earlier observations that inhibitors of ACE—or kininase II—can protect against cardiac ischemia even in isolated, buffer-perfused rat hearts. Bradykinin antagonism largely abolishes the protective effects of an ACE inhibitor in isolated hearts. Furthermore, bradykinin seems to be generated locally even in isolated buffer-perfused hearts. This suggests that accumulation of bradykinin may be an important mechanism by which ACE inhibitors may protect the heart against ischemic damage.

However, it is not yet established whether increased bradykinin is sufficient to protect against cardiac ischemia.

Therefore, we used a transgenic rat model we recently developed, where the entire human kallikrein gene (hKLK1) was introduced under the control of the heavy metal-responsive mouse metallothionein (mMT1) promoter into a Sprague-Dawley background.

To study the effects of regional cardiac ischemia in isolated perfused hearts, the left coronary artery was reversibly occluded for 15 min in 20 transgenic and 20 transgene negative littermate controls. The hearts were divided to receive either an intracoronary infusion of the bradykinin B2 receptor antagonist HOE140 (10^-9 M) or vehicle during the experiment.

Total overflow of purines (adenosine breakdown products inosine, hypoxanthine, and xanthine) in the coronary effluent and the first derivative of left ventricular pressure (systolic dP/dt) were measured at various times, as indicators of ischemic damage and of the effects of ischemia on left ventricular function.

PRINCIPAL FINDINGS

1. General characteristics
In vivo hemodynamic parameters and parameters of left ventricular function were similar in both strains. Measurement of LVP and dP/dt in vivo revealed no differences between the strains and demonstrated a remarkable similarity between dP/dt measured in vivo or ex vivo.

2. Ischemia reperfusion in the isolated heart
During ischemia, flow decreased to a similar extent in all groups, irrespective of transgene status or of HOE140 administration, and postischemic hyperemia occurred in all four groups to a similar extent. However, the total amount of purines released upon reperfusion was significantly less in the untreated transgenic rats, as compared to the untreated nontransgenic control littermates. HOE140 abolished this difference. HOE140 did not have an effect in control rats on the total overflow of purines (see Fig. 1 ).

View larger version (23K): [in this window] [in a new window]   Figure 1. The effects of intracoronary infusion of HOE140 and the effects of the transgene status are visualized by depicting two of the four groups at a time. A, B) The differences between the Sprague-Dawley (SD) transgene negative littermates and the transgenic rats (TGR) are depicted after vehicle treatment (A) or after intracoronary B2 receptor antagonist HOE140 (B). C, D) The effects of HOE140 are depicted in the SD transgene negative littermates, or in the transgenic rats (D). The left coronary artery was occluded from time 15 min until 30 min, whereupon the region was reperfused. Overflow of total purines upon reperfusion is depicted for the four groups. Total purine clearly peaks upon reperfusion, reflecting the washout of ischemia induced breakdown products. HOE140 itself did not affect the overflow of purines, as in the transgene negative littermates HOE140 did not alter the level of purines (C). However, in the untreated transgenic rats, the overflow of purines was significantly reduced (A), which was abolished by coinfusion of HOE140 (B).

Furthermore, although systolic dP/dt clearly decreased during left ventricular ischemia, dP/dt decreased significantly less in the transgenic rats, a difference that was abolished by coinfusion of HOE140 (see Fig. 2 ).

View larger version (26K): [in this window] [in a new window]   Figure 2. Changes in systolic dP/dt as a measure of left ventricular function are depicted for the four groups. Systolic dP/dt clearly decreases upon the start of ischemia and recovers after releasing the ligature. This decrease in dP/dt was significantly less in the untreated transgenic rats (A). This difference was abolished by coinfusion of HOE140 (B). HOE140 itself decreased systolic dP/dt in both transgenic and SD control rats (C, D) before the start of ischemia, although this did not attain statistical significance in either group.

DISCUSSION

In this study we addressed in a transgenic rat model whether a chronic, primary increase of kallikrein protects against cardiac ischemia. We show that cardiac ischemia-reperfusion injury is attenuated in the transgenic rats that overexpress kallikrein, as demonstrated by both decreased formation of nucleotide breakdown products and attenuated impairment of systolic contractility after cardiac ischemia in the isolated heart of these transgenic rats. The B2 specific antagonist HOE140, when given acutely in the isolated heart, completely abolished these protective effects. Taken together, this demonstrates 1) that a chronic increase in bradykinin is sufficient to protect the heart against ischemic damage and 2) that this is due to an acute effect of bradykinin. This does not seem due to properties of HOE140 itself, since HOE140 did not alter ischemic damage in normal control rat hearts.

The importance of this finding is that for the first time it is clear that an increase in bradykinin is sufficient to protect against ischemic damage. Since the first description that ACE inhibitors protect against ischemic damage even in an isolated, buffer-perfused rat heart, it has been suggested that bradykinin may be instrumental in this protection. Several studies have indeed shown that HOE140 could counteract these ex vivo effects of ACE inhibitors, which substantiates a role for bradykinin. Our study demonstrates that even the effects of a long-standing high kallikrein expression could be acutely counteracted by HOE140 when given only ex vivo just minutes prior to the start of ischemia. This negates the option that long-standing effects of increased bradykinin,via structural alterations, would confer the protective effects of high kallikrein expression. The mechanism whereby increased bradykinin protects against ischemia remains ill understood. The putative mechanisms can now be narrowed down to those that act acutely, such as ischemic preconditioning (see diagram). In accordance, recent clinical studies also suggest an immediate protective effect of local bradykinin in patients undergoing percutaneous coronary balloon angioplasty.

The protection against ischemic damage in the transgenic rats was also evidenced by an attenuated impairment of systolic dP/dt.

In conclusion, we demonstrate for the first time that increased bradykinin is sufficient to protect against cardiac ischemic damage and that this effect is due to an acute protective mechanism against myocardial injury, possibly via ischemic preconditioning.

View larger version (22K): [in this window] [in a new window]   Scheme 1. No caption available.

ACKNOWLEDGMENTS

This work was supported in part by European Community grant no: ERBCHGCT 940725 and by an ICIN Molecular Cardiology fellowship to Y.M.P.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.99-1011fje

This Article Full Text (PDF) All Versions of this Article: 14/13/1861 99-1011fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by PINTO, Y. M. Articles by BUIKEMA, H. Search for Related Content PubMed PubMed Citation Articles by PINTO, Y. M. Articles by BUIKEMA, H.