Steffen Sandmann

Transcriptional and translational regulation of calpain in the rat heart after myocardial infarction – effects of AT1 and AT2 receptor antagonists and ACE inhibitor

Recent studies demonstrated that the cardiac calpain system is activated during ischaemic events and is involved in cardiomyocyte injury. The aim of this study was to investigate the contribution of AT1 and AT2 receptors in the regulation of calpain‐mediated myocardial damage following myocardial infarction (MI). Infarcted animals were treated either with placebo, the ACE inhibitor ramipril (1 mg kg−1 d−1), the AT1 receptor antagonist valsartan (10 mg kg−1 d−1) or the AT2 receptor antagonist PD 123319 (30 mg kg−1 d−1). Treatment was started 7 days prior to surgery. On day 1, 3, 7 and 14 after MI, gene expression and protein levels of calpain I, II and calpastatin were determined in left ventricular free wall (LVFW) and interventricular septum (IS). At day 3 and 14 post MI, morphological investigations were performed. Calpain I mRNA expression and protein levels were increased in IS 14 days post MI, whereas mRNA expression and protein levels of calpain II were maximally increased in LVFW 3 days post MI. Ramipril and valsartan decreased mRNA and protein up‐regulation of calpain I and II, and reduced infarct size and interstitial fibrosis. PD 123319 did not affect calpain I or II up‐regulation in the infarcted myocardium, but decreased interstitial fibrosis. Calpastatin expression and translation were not affected by AT receptor antagonists or ACE inhibitor. Our data demonstrate a distinct, temporary‐spatial up‐regulation of calpain I and II following MI confer with the hypothesis of calpain I being involved in cardiac remodelling in the late and calpain II contributing to cardiac tissue damage in the early phase of MI. The up‐regulation of calpain I and II is partly mediated via the AT1 receptor and can be reduced by ACE inhibitors and AT1 receptor antagonists.

Differential effects of angiotensin AT1and AT2receptors on the expression, translation and function of the Na+-H+exchanger and Na+-HCO3−symporter in the rat heart after myocardial infarction

OBJECTIVES This study investigated the role of angiotensin receptor subtype 1 (AT1) and angiotensin receptor subtype 2 (AT2) in the regulation of Na+-H+ exchanger (NHE) and Na+-HCO3 symporter (NBC) in the infarcted myocardium. BACKGROUND The cardiac renin-angiotensin system is activated after myocardial infarction (MI), and both angiotensin AT1 and AT2 receptors are upregulated in the myocardium. METHODS Na+-H+ exchanger isoform-1 and NBC-1 gene expression were determined by reverse transcription polymerase chain reaction and Northern blot analysis; protein levels by Western blot analysis; and activity by measurement of H+ transport in left ventricular (LV) free wall, interventricular septum (IS) and right ventricle (RV) after induction of MI. Rats were treated with placebo, the angiotensin-converting enzyme inhibitor ramipril (1 mg/kg/day), the AT1 receptor antagonist valsartan (10 mg/kg/day) or the AT2 receptor antagonist PD 123319 (30 mg/kg/day). Treatment was started seven days before surgery. RESULTS Na+-H+ exchanger isoform-1 and NBC-1 messenger RNA (mRNA) expression and protein levels were increased twofold in the LV free wall after MI, whereas no changes were observed in the IS and RV. Na+-dependent H+ flux was increased in the LV free wall. Ramipril inhibited mRNA and protein upregulation of both transporters. Valsartan inhibited the upregulation of NHE-1 mRNA and protein but had no effect on NBC-1 mRNA expression and translation. In contrast, PD 123319 abolished the upregulation of NBC-1 mRNA and protein but had no effect on NHE-1 upregulation. Ramipril and valsartan prevented post-MI increase in NHE-1 activity, whereas ramipril and PD 123319 decreased NBC-1 activity. CONCLUSIONS Angiotensin II via its AT1 and AT2 receptors differentially controls transcriptional and translational regulation as well as the activity of NHE-1 and NBC-1 in the ischemic myocardium and contributes to the control of pH regulation in cardiac tissue.

Activity profile of calpains I and II in chronically infarcted rat myocardium – influence of the calpain inhibitor CAL 9961

The calpains have been proposed to be activated following cardiac ischaemia and to contribute to myocyte damage after myocardial infarction (MI). In this study, the activity of calpains I and II in the infarcted and non‐infarcted rat myocardium and the action of the selective calpain inhibitor, CAL 9961, has been investigated. MI was induced by permanent ligation of the left coronary artery. One, 3, 7 and 14 days post MI, the enzymes calpain I and II were separated from homogenates of the interventricular septum (IS) and left ventricular free wall (LVFW) by chromatography on DEAE‐Sepharose. The activity of the calpains was measured in sham‐operated and MI animals chronically treated with placebo or CAL 9961 (15 mg kg−1 d−1 s.c.) in a synthetic substrate assay. Treatment was started 3 days before MI induction. Calpain I activity reached highest values in IS 14 days post MI, whereas maximum activity of calpain II was measured in LVFW 3 days post MI. In experiments in vitro, CAL 9961 completely inhibited both calpains. In vivo, chronic treatment of MI animals with CAL 9961 partially prevented the increase in calpain I activity in IS and reduced calpain II activity in LVFW to sham levels. Our findings demonstrate that calpains I and II are activated after MI, however, both enzymes differ in their regional and temporal activation within the infarcted myocardium. Chronic inhibition of these enzymes with CAL 9961 might limit the calpain‐induced myocardial damage and preserve cardiac structural integrity post MI.

Cardioprotective effects of the Na+/H+-exchange inhibitor cariporide in infarct-induced heart failure

OBJECTIVE We investigated the effect of chronic treatment with the new Na(+)/H(+)-exchange inhibitor, cariporide, on cardiac function and remodelling 6 weeks after myocardial infarction (MI) in rats. METHODS Treatment with cariporide was commenced either 1 week pre or 30 min, 3 h, 24 h or 7 days after ligation of the left ventricular artery and was continued until haemodynamic parameters were obtained 6 weeks after MI in conscious rats. RESULTS Compared to sham animals, untreated MI-controls developed pronounced heart failure after 6 weeks. Basal left ventricular end-diastolic pressure (in mmHg) was reduced in the groups in which cariporide was started 1 week pre (16.0+/-1.7) or 30 min (12.5+/-1.1), 3 h (11.8+/-1.0) and 24 h (13.0+/-2.5) after MI compared to untreated MI-controls (22. 4+/-1.5; P<0.01). Basal myocardial contractility (in 1000 mmHg/s) was only increased when treatment was initiated after 30 min (9. 0+/-0.7), 3 h (8.5+/-0.3) and 24 h (8.0+/-0.7) compared to untreated MI-controls (5.8+/-0.7; P<0.05-0.01). Infarct size (in % of left ventricular circumference) was 40.0+/-2.1 in MI-controls and was decreased when treatment was begun after 30 min (32.6+/-2.7) or 3 h (32.4+/-2.3) (P<0.05). In animals, in which cariporide was started 3 h after induction of MI, heart weight/body weight ratio was significantly decreased, indicating reduced cardiac hypertrophy. When treatment started 7 days after MI, cariporide did not exert any beneficial actions on structural and functional cardiac parameters. CONCLUSION Our results show for the first time that chronic treatment with the Na(+)/H(+)-exchange inhibitor cariporide engendered marked cardioprotective effects when commenced before and up to 24 h after MI. The optimal time for the start of treatment was between 30 min and 3 h post MI.

Calcium channel blockade limits cardiac remodeling and improves cardiac function in myocardial infarction-induced heart failure in rats.

Calcium channel antagonists (CCAs) have been proposed to prevent cardiac events after myocardial infarction (MI). However, unwanted effects, such as negative inotropy, limit their use in many cases. The aim of this study was to compare the effects of long-term treatment with the CCAs, mibefradil, verapamil, and amlodipine, administered before and after chronic MI on myocardial remodeling and cardiac function. MI was induced by permanent ligation of the left coronary artery in male Wistar rats. Infarcted animals were treated with placebo, mibefradil (10 mg/kg/d po), verapamil (8 mg/kg bid po), or amlodipine (4 mg/kg/d po). Treatment was started 7 days before or 3 h after MI induction. Six weeks after MI, mean arterial blood pressure (MAP), heart rate (HR), left ventricular end diastolic pressure (LVEDP), and cardiac contractility (dP/dtmax) were measured. Morphometric parameters such as infarct size (IS), left ventricular dilation (LVD), septal thickness (ST), and cardiac fibrosis were determined in picrosirius red-stained hearts. Six weeks after MI, MAP and dP/dtmax were decreased, whereas LVEDP and HR were increased in placebo-treated controls. The hearts featured an IS of 45%, left ventricular dilation, cardiac fibrosis, and septal thinning. MAP of all CCA-treated animals was increased, whereas LVEDP was decreased and dP/dtmax increased 7-day pre- and 3-h post-MI started in mibefradil- and amlodipine-treated animals, but not in verapamil-treated animals. In contrast to amlodipine treatment, before and after MI started mibefradil and verapamil treatment decreased HR. Pretreatment with all CCA reduced IS and increased ST, whereas only mibefradil and amlodipine pretreatment prevented LVD and cardiac fibrosis. After MI started treatment with mibefradil and amlodipine reduced IS and cardiac fibrosis, and increased ST. Long-term treatment with the CCAs mibefradil, verapamil, and amlodipine reduced myocardial remodeling and improved cardiac function in MI-induced heart failure in rats.

Effects of the calcium channel antagonist mibefradil on haemodynamic parameters and myocardial Ca2+-handling in infarct-induced heart failure in rats

Objective: Abnormal intracellular Ca2+-handling has been implicated in the pathogenesis of contractile dysfunction and arrhythmias in failing hearts. Calcium channel antagonists (CCA) have been proposed for the prevention of cardiac events after myocardial infarction (MI). Recent studies suggest that the blockade of T-type Ca2+-channels induced a heart rate reduction without negative inotropic effects. We investigated the effects of the preferentially T-channel blocking CCA, mibefradil, on haemodynamic parameters and intramyocardial Ca2+-handling and contractility in the early and late period after MI. Methods: MI was induced by permanent ligation of the left coronary artery in male normotensive Wistar rats. Animals were divided in sham-operated and placebo- or mibefradil-treated MI rats. Placebo or Mibefradil treatment (10 mg/kg/d via gastric gavage) was started 7 days prior to MI-induction. Haemodynamic and intramyocardial Ca2+ measurements were performed 1, 3, 7 and 42 days after surgery. At these time points, mean arterial blood pressure (MAP), heart rate (HR), left ventricular enddiastolic pressure (LVEDP) and cardiac contractility (d P /d t max) were measured in conscious rats. After haemodynamic measurements, the left ventricular papillary muscle was separated to determine developed tension (DT), time to peak tension (TPT) and systolic and diastolic free intracellular Ca2+ concentrations ([Ca2+]i) using the Ca2+ indicator aequorin. Dose-response curves after extracellular isoproterenol- or Ca2+-stimulation were recorded. Results: In the early (1–3 days) period after MI, MAP and d P /d t max were decreased and LVEDP and HR were increased in placebo-treated MI rats. Mibefradil treatment increased MAP and d P /d t max and decreased LVEDP and HR in infarcted rats. In the papillary muscle of placebo-treated rats, MI induced a decrease in DT and an increase in TPT and in diastolic and systolic [Ca2+]i. DT of placebo-treated MI rats showed a reduced reactivity after isoproterenol- or Ca2+-stimulation. After mibefradil treatment DT was increased and TPT was reduced in the late period (7–42 days) after MI, and diastolic and systolic [Ca2+]i were decreased in the early period after MI (1–3 days). The inotropic response to β-adrenergic or extracellular Ca2+-stimulation was markedly improved by mibefradil 7 and 42 days after MI. Conclusion: We conclude, that mibefradil improves cardiac function, protects the myocardium against ischemia-induced Ca2+-overload and increases β-adrenergic responsiveness in chronically failing rat hearts.

Calcium channel blockade limits transcriptional, translational and functional up-regulation of the cardiac calpain system after myocardial infarction.

Abnormal Ca(2+) inward current through cardiac Ca(2+) channels during ischemia has been shown to be an initial signal for activation of myocardial Ca(2+)-dependent enzymes. This study investigated the contribution of cardiac L- and T-type Ca(2+) channels in the calpain-mediated myocardial damage following myocardial infarction. Myocardial infarction was induced by permanent ligation of the left coronary artery. Infarcted rats were orally treated with placebo, amlodipine (L-channel blockade; 4 mg/kg/day) or mibefradil (L-/T-channel blockade; 10 mg/kg/day) beginning 7 days before induction of myocardial infarction. Gene expression, protein levels and enzyme activity of calpains I and II were measured 1, 3, 7 and 14 days postcoronary occlusion in the noninfarcted and infarcted myocardium. Infarct size, left ventricular dilation and interstitial collagen volume fraction were determined in picrosirius red-stained hearts. Myocardial infarction induced an up-regulation of calpain I mRNA, protein and activity in the noninfarcted myocardium (maximum 14 days postinfarction), whereas mRNA, protein and activity of calpain II were maximally increased in the infarcted myocardium 3 days postinfarction. Fourteen days postinfarction, infarct size was 49%, the left ventricle was dilated and interstitial collagen volume fraction was increased. Amlodipine-inhibited mRNA, protein and activity up-regulation of calpain I decreased interstitial collagen volume fraction and infarct size. Mibefradil-attenuated mRNA, protein and activity up-regulation of calpain II at all four time points measured and of calpain I at 7 and 14 days postinfarction reduced infarct size and prevented left ventricular dilation. Infarction-induced cardiac hypertrophy was accompanied by an up-regulation of calpain I, whereas calpain II was up-regulated in the infarcted myocardium. Cardiac L- and T-type Ca(2+) channel blockade differentially reduced postinfarction remodeling associated with selective inhibition of cardiac calpains I and II, respectively.

Bradykinin B1 and B2 receptors differentially regulate cardiac Na+-H+ exchanger, Na+-Ca2+ exchanger and Na+-HCO3- symporter.

Bradykinin B(1) and B(2) receptors are up-regulated in the infarcted myocardium, and both receptors are involved in the regulation of intracellular pH and Ca(2+). The present study investigated the role of bradykinin B(1) and B(2) receptors in the regulation of Na(+)-H(+) exchanger (NHE-1), Na(+)-Ca(2+) exchanger (NCE-1) and Na(+)-HCO(3)(-) symporter (NBC-1) in the infarcted myocardium. NHE-1, NCE-1 and NBC-1 mRNA expression was determined by Northern blot analysis and the protein levels by Western blot analysis. Measurements were performed 1, 7 and 14 days after induction of myocardial infarction. Localization of NHE-1, NCE-1 and NBC-1 within the myocardium was studied using confocal microscopy. Cardiac morphology was measured in picrosiris-red-stained hearts. Rats were treated with placebo, the bradykinin B(2) receptor antagonist icatibant (0.5 mg/kg/day) or the bradykinin B(1) receptor antagonist des-Arg(9)-[Leu(8)]bradykinin (1 mg/kg/day). Treatment was started 1 week prior to surgery and continued until 1, 7 and 14 days post infarction. NHE-1, NCE-1 and NBC-1 mRNA expression and protein levels were increased 1 day and reached maximum values on day 7 post infarction. NHE-1 was localized in the plasma membrane, NCE-1 in the membrane of the sarcoplasmatic reticulum and NBC-1 near the Z-line. Icatibant reduced NHE-1 and inhibited NCE-1 mRNA- and protein up-regulation, while des-Arg(9)-[Leu(8)]bradykinin had no effect on NHE-1 and NCE-1 expression and translation. Transcriptional and translational up-regulation of NBC-1 was unaffected by the bradykinin B(1) and B(2) receptor antagonists. Icatibant, but not des-Arg(9)-[Leu(8)]bradykinin, limited infarct size and reduced left ventricular dilation, septal thickening and interstitial fibrosis post infarction. Bradykinin B(2) receptors are involved in transcriptional and translational regulation of NHE-1 and NCE-1 in the ischemic myocardium. Chronic B(2) receptor blockade might exert an anti-ischemic effect via limitation of NHE-1-mediated acidosis and NCE-1-mediated Ca(2+)-overload.