Chronic dual inhibition of angiotensin-converting enzyme and neutral endopeptidase during the development of left ventricular dysfunction in dogs.

Angiotensin-converting enzyme (ACE) inhibition as well as neutral endopeptidase (NEP) inhibition was demonstrated to influence hemodynamics in various cardiac disease states. However, specific effects of chronic combined ACE and NEP inhibition on left ventricular (LV) and myocyte geometry and function remain unclear. In this study, a dual-acting metalloprotease inhibitor (DMPI), which possesses both ACE and NEP inhibitory activity, was used in a rapid-pacing model of LV dysfunction. LV and myocyte geometry and function were examined in control dogs (n = 6), in dogs with pacing-induced LV dysfunction (216 +/- 2 beats/min, 28 days, n = 7), and in dogs with DMPI treatment during rapid pacing (10 mg/kg p.o., b.i.d., n = 6). With chronic rapid pacing, LV end-diastolic volume increased (84 +/- 4 vs. 49 +/- 3 ml), and LV ejection fraction decreased (38 +/- 3% vs. 68 +/- 3%) compared with control (p < 0.05). DMPI concomitantly administered during long-term rapid pacing did not change LV ejection fraction (35 +/- 3%), but LV end-diastolic volume was reduced (70 +/- 5 vs. 84 +/- 4 ml; p < 0.05) when compared with rapid pacing only. With long-term rapid pacing, myocyte cross-sectional area was decreased (278 +/- 5 vs. 325 +/- 5 microm2), and resting length increased (178 +/- 2 vs. 152 +/- 1 microm) when compared with control (p < 0.05). With DMPI concomitantly administered during rapid pacing, myocyte cross-sectional area (251 +/- 5 microm2) and resting length (159 +/- 4 microm) were reduced when compared with rapid pacing only (p < 0.05). Myocyte velocity of shortening decreased from control values with long-term rapid pacing (39.3 +/- 3.9 vs. 73.2 +/- 5.9 microm/s; p < 0.05) but improved with DMPI treatment during rapid pacing when compared with rapid pacing only (58.9 +/- 6.7 microm/s; p < 0.05). Myocyte velocity of shortening with beta-adrenergic-receptor stimulation (25 nM isoproterenol) was reduced from controls with rapid pacing (125 +/- 12 vs. 214 +/- 30 microm/s; p < 0.05) but was improved with DMPI treatment during rapid pacing when compared with rapid pacing only (178 +/- 12 microm/s; p < 0.05). In a model of rapid pacing-induced LV failure, concomitant DMPI treatment significantly reduced the degree of LV dilation with no apparent effect on LV pump function. At the level of the LV myocyte, long-term DMPI treatment with rapid pacing improved myocyte performance and beta-adrenergic response. Thus the improvement in isolated myocyte contractile function was not translated into improved global LV-pump performance. The mechanisms by which improved myocyte contractility was not translated into a beneficial effect on LV-pump function with DMPI treatment during rapid pacing remain speculative, but likely include significant changes in LV remodeling and loading conditions.

Angiotensin-converting enzyme inhibition and angiotensin II subtype-1 receptor blockade during the progression of left ventricular dysfunction: differential effects on myocyte contractile processes.

Inhibition of the angiotensin-converting enzyme (ACE) in the setting of chronic left ventricular (LV) dysfunction has been demonstrated to have beneficial effects on survival and symptoms. However, whether ACE inhibition has direct effects on myocyte contractile processes and if these effects are mediated primarily through the AT1 angiotensin-II receptor subtype remains unclear. The present project examined the relationship between changes in LV and myocyte function and beta adrenergic receptor transduction in four groups of six dogs each: (1) Rapid Pace: LV failure induced by chronic rapid pacing (4 weeks; 216 +/- 2 bpm); (2) Rapid Pace/ACEI: concomitant ACE inhibition (ACEI: fosinopril 30 mg/kg b.i.d.) with chronic pacing; (3) Rapid Pace/AT1 Block: concomitant AT1 Ang-II receptor blockade [Irbesartan: SR 47436(BMS-186295) 30 mg/kg b.i.d.] with chronic pacing; and (4) CONTROL: sham controls. With Rapid Pace, the LV end-diastolic volume increased by 62% and the ejection fraction decreased by 53% from control. With Rapid Pace/ACEI, the LV end-diastolic volume was reduced by 24% and the ejection fraction increased by 26% from Rapid Pace only values. Rapid Pace/AT1 Block did not improve LV geometry or function from Rapid Pace values. Myocyte contractile function decreased by 40% with Rapid Pace and increased from this value by 32% with Rapid Pace/ACEI. Rapid Pace/AT1 Block had no effect on myocyte function when compared with Rapid Pace values. With Rapid Pace/ACEI, beta receptor density and cyclic AMP production were normalized and associated with an improvement in myocyte beta adrenergic response compared with Rapid Pace only. Although Rapid Pace/AT1 also normalized beta receptor density, cyclic AMP production was unchanged and myocyte beta adrenergic response was reduced by 15% compared with Rapid Pace only. ACE inhibition with chronic rapid pacing improved LV and myocyte geometry and function, and normalized beta receptor density and cyclic AMP production. However, AT1 Ang-II receptor blockade with chronic rapid pacing failed to provide similar protective effects on LV and myocyte geometry and function. These unique findings suggest that the effects of ACE inhibition on LV geometry and myocyte contractile processes in the setting of developing LV failure are not primarily caused by modulation of AT1 Ang-II receptor activation.

Direct effects of protamine sulfate on myocyte contractile processes. Cellular and molecular mechanisms.

BACKGROUND: Administration of the arginine-rich, highly charged protamine (PROT) molecule has been associated with episodes of acute left ventricular (LV) dysfunction. The objective of the present study was to test the hypothesis that PROT has direct effects on isolated LV myocyte contractile processes and sarcolemmal transduction systems. METHODS AND RESULTS: Exposure of porcine LV myocytes (n = 305) to 40 micrograms/mL PROT (reflecting a dose of 2.5 mg/kg) decreased basal contractile function and beta-adrenergic responsiveness. For example, myocyte percent shortening was 4.3 +/- 0.1% in control myocytes and decreased to 2.8 +/- 0.2% in the presence of 40 micrograms/mL PROT (P < .05). Myocyte percent shortening was 9.3 +/- 0.7% after beta-adrenergic receptor stimulation (isoproterenol; 25 nmol/L) and was significantly reduced in the presence of 40 micrograms/mL PROT (5.7 +/- 0.7%, P < .05). PROT reduced myocyte responsiveness to forskolin (100 mumol/L), which directly activates adenylate cyclase, by > 40% from forskolin. In addition, PROT abolished the inotropic effects of ouabain on myocyte contractile function. To determine contributory mechanisms for the effects of PROT on myocyte sarcolemmal systems, beta-receptor- and cardiac glycoside-binding characteristics were determined in sarcolemmal preparations. beta-receptor binding was 175 +/- 10 fmol/mg and was reduced to 140 +/- 6 fmol/mg in the presence of PROT (P < .05). Ouabain receptor binding was 7.1 pmol/mg and decreased to 2.6 +/- 0.4 pmol/mg in the presence of PROT. In addition, cAMP production after stimulation with isoproterenol and forskolin was significantly blunted in the presence of PROT. Variants of the PROT moelcule were constructed by specific amino acid substitutions and deletions, which provided a means to vary charge as well as structure. Substitution of arginine with lysine in the PROT peptide sequence ameliorated the negative effects on myocyte contractile processes; despite identical overall charge (21+). However, a PROT variant with an 18+ charge but different amino acid sequence induced significant negative effects on myocyte function and inotropic responsiveness. Thus, the effects of PROT on myocyte contractile processes are not due simply to the high positive charge of the molecule. To further establish that PROT can contribute to changes in LV function in the clinical setting, fluorescein-labeled PROT was circulated in antegradely perfused rabbit hearts. Microscopic examination revealed that PROT could traverse the vascular compartment of the myocardium and come in direct contact with the myocyte. CONCLUSIONS: The unique findings from the present study suggest that a fundamental contributory mechanisms for the changes in LV function observed after protamine administration may be the direct effect of unbound protamine on myocyte contractile processes.

Angiotensin-converting enzyme inhibition and the progression of congestive cardiomyopathy. Effects on left ventricular and myocyte structure and function.

BACKGROUND: Clinical trials have demonstrated that angiotensin-converting enzyme inhibition (ACEI) improves survival in patients with long-term left ventricular (LV) dysfunction. However, it remained unclear from these clinical reports whether the beneficial effects of ACEI were due to direct improvements in LV myocardial structure and function. Accordingly, the overall objective of the present study was to examine the direct effects of ACEI on both LV and myocyte structure and function in the setting of cardiomyopathic disease. METHODS AND RESULTS: LV and isolated myocyte function and structure were examined in control dogs (n = 6), in dogs after the development of dilated cardiomyopathy caused by rapid ventricular pacing (RVP, 216 beats per minute, 4 weeks, n = 6), and in dogs with RVP and concomitant ACEI (RVP/ACEI, fosinopril 30 mg/kg BID, n = 6). LV ejection fraction fell with RVP compared with control values (35 +/- 3 versus 73 +/- 2%, P < .05) and was higher with RVP/ACEI compared with RVP values (41 +/- 4%, P = .048). LV end-diastolic volume increased with RVP compared with control values (78 +/- 7 versus 101 +/- 7 cm3, P < .05) and was lower with RVP/ACEI (82 +/- 3 cm3, P < .05). Isolated myocyte length increased with RVP (182 +- 1 versus 149 +/- 1 micron), and the velocity of shortening decreased (36 +/- 1 versus 57 +/- 1 micron/s) compared with control values (P < .05). With RVP/ACEI, myocyte length was reduced (169 +/- 1 micron) and velocity of shortening was increased (45 +/- 1 micron/s) compared with RVP values (P < .05). Myocyte velocity of shortening after beta-adrenergic receptor stimulation with 25 nmol/L isoproterenol was reduced with RVP compared with control values (142 +/- 5 versus 193 +/- 8 micron/s, P < .05) and significantly improved with RVP/ACEI (166 +/- 6 micron/s, P < .05). In the RVP group, beta-adrenergic receptor density fell 26%, and cAMP production with beta-adrenergic receptor stimulation was reduced 48% from control values. RVP/ACEI resulted in a normalization of beta-adrenergic receptor density and cAMP production. LV myosin heavy-chain content when normalized to dry weight of myocardium was unchanged with RVP (149 +/- 11 mg per gram dry weight of myocardium [gdwt]) and RVP/ACEI (150 +/- 4 mg/gdwt) compared with control values (165 +/- 4 mg/gdwt). LV collagen content decreased with RVP compared with control values (7.6 +/- 0.4 versus 9.6 +/- 0.8 mg per gram wet weight of myocardium [gwwt], P < .05) but was increased with RVP/ACEI (14.4 +/- 1.3 mg/gwwt, P < .05). CONCLUSIONS: Concomitant ACEI with chronic tachycardia reduced LV chamber dilation and improved myocyte contractile function and beta-adrenergic responsiveness. Contributory cellular and extracellular mechanisms for the beneficial effects of ACEI in this model of dilated cardiomyopathy included a normalization of beta-adrenergic receptor function and enhanced myocardial collagen support. The results from this study provide evidence that ACEI during the development of cardiomyopathic disease provided beneficial effects on LV myocyte contractile processes and myocardial structure.

Differential effects of protamine sulfate on myocyte contractile function with left ventricular failure.

OBJECTIVES: This project tested two fundamental hypotheses: 1) Protamine sulfate has a direct and negative effect on myocyte contractile processes; 2) the negative effects of protamine on myocyte contractility will be enhanced in the setting of chronic left ventricular dysfunction. BACKGROUND: An increasing number of patients undergoing cardiac and vascular surgical procedures have underlying chronic left ventricular dysfunction. Protamine sulfate is commonly required during these surgical procedures but has been associated with left ventricular dysfunction. However, it is not known whether protamine may have a direct and selective effect on myocyte contractility in the setting of chronic left ventricular dysfunction. METHODS: This study examined the direct effects of protamine on isolated myocyte contractile function in 10 control pigs and 10 pigs with dilated cardiomyopathy induced by supraventricular tachycardia (rapid atrial pacing at 240 beats/min for 3 weeks). Myocyte contractile function was measured by videomicroscopy at baseline and with 10, 20, 40 or 80 micrograms/ml of protamine. In a second series of experiments, myocytes were preincubated with protamine and then stimulated with the beta-adrenergic agonist isoproterenol (25 nmol/liter). RESULTS: In the presence of 20 micrograms/ml of protamine, myocyte contractile function was unaffected in the control group but decreased by 40% from baseline values in the supraventricular tachycardia group. With 10 micrograms/ml of protamine, myocyte beta-adrenergic responsiveness was reduced by 25% in the supraventricular tachycardia group with no change in the control group. In the presence of 40 and 80 micrograms/ml of protamine, myocyte contractile function decreased in both groups. However, 40 micrograms/ml of protamine caused a more pronounced decline in myocyte function and beta-adrenergic responsiveness in the supraventricular tachycardia group. CONCLUSIONS: An increased sensitivity to the depressive effects of protamine on myocyte contractile function and beta-adrenergic responsiveness occurred in this model of chronic left ventricular dysfunction. These results suggest that patients with underlying cardiac disease may have an increased susceptibility to a sudden compromise of left ventricular contractile performance after protamine administration.

Direct effects of thrombin on myocyte contractile function.

Cardiopulmonary bypass activates the clotting cascade, resulting in elevated circulating levels of thrombin. In light of the fact that the function of a wide variety of cell types is modulated by thrombin, we hypothesized that thrombin may have a direct effect on myocyte function. Isolated left ventricular myocyte contractile function was measured from 6 adult dogs using videomicroscopy at baseline and after increasing concentrations of thrombin (1 to 10 U/mL). Indices of myocyte contractile function were reduced in a dose-dependent manner in the presence of increasing concentrations of thrombin. For example, myocyte percent shortening fell by 18% with 1 U/mL thrombin and by 43% with 2 U/mL thrombin. The addition of hirudin, a highly selective thrombin inhibitor, completely blocked the effects of thrombin on myocyte contractile function. beta-Adrenergic agonists are commonly used in the early post-cardiopulmonary bypass period. Accordingly, a final set of experiments examined the effects of thrombin on myocyte beta-adrenergic responsiveness using isoproterenol (25 nmol/L). In myocytes preincubated with 1 U/mL thrombin, myocyte beta-adrenergic responsiveness was significantly reduced. For example, in the presence of 1 U/mL thrombin, myocyte velocity of shortening fell by 25% from isoproterenol alone values. The results from the present study provide evidence that thrombin has a direct negative effect on steady-state contractile function and beta-adrenergic responsiveness in adult mammalian ventricular myocytes. These findings suggest that thrombin may be an additional contributory factor toward the transient left ventricular dysfunction that has been observed after cardiopulmonary bypass.

The direct effects of protamine sulfate on myocyte contractile processes.

The use of protamine sulfate in patients has been associated with circulatory collapse and is suspected to directly depress left ventricular function. However, the cellular basis for these changes that occur after protamine administration are unknown. Accordingly, the first objective of this study was to determine the direct effects of protamine on isolated myocyte contractile function. Myocytes were isolated from porcine hearts and contractile function was examined at baseline and then after the administration of protamine in concentrations of 20, 40, or 80 micrograms/ml. These concentrations were chosen because they reflect the serum concentrations of protamine commonly obtained in patients. The presence of protamine resulted in a dose-dependent decline in myocyte contractile function. For example, in the presence of a 20 microgram/ml concentration of protamine myocyte contractile function did not change significantly from baseline values, whereas an 80 microgram/ml protamine concentration caused myocyte percent and velocity of shortening to fall by more than 35% from baseline values. In light of the fact that protamine directly depressed myocyte contractile function, a second objective of this study was to examine potential cellular mechanisms responsible for this effect. Accordingly, in the next series of experiments, the effects of protamine on the myocyte sarcolemmal beta-adrenergic receptor system were examined by measuring myocyte contractile function with the beta-adrenergic agonist isoproterenol (25 nmol/L), as well as with the concomitant addition of protamine and isoproterenol. In the presence of protamine, myocyte beta-adrenergic responsiveness was significantly reduced. For example, in the presence of an 80 microgram/ml dose of protamine, both myocyte percent and velocity of shortening fell by greater than 50% when compared with isoproterenol alone values (p < 0.05). To determine the reversibility of these protamine effects, we performed additional experiments in the presence of heparin. Incubation with heparin before protamine addition prevented the negative effects of protamine on myocyte function. However, the addition of heparin after protamine incubation failed to reverse the negative effects of protamine on myocyte function. In a final set of experiments, the effects of protamine on isolated myocyte electrophysiologic properties were examined using microelectrode techniques at baseline and with either 40 or 80 micrograms/ml doses of protamine. Myocyte resting membrane potential changed from baseline with the addition of a 40 micrograms/ml dose of protamine (-79.2 +/- 0.5 versus -75.2 +/- 0.8 mV (p < 0.05), with no further change at an 80 micrograms/ml dose of protamine (-73.0 +/- 1.3 mV).(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of protamine on myocyte contractile function and beta-adrenergic responsiveness.

The use of protamine sulfate in patients has been associated with severe circulatory collapse and myocardial failure. However, the exact mechanisms responsible for these reactions to protamine remain unclear. Accordingly, we examined the effect of protamine on isolated myocyte contractile function. Indexes of isolated myocyte contractile function, percent shortening, and velocity of shortening were examined using videomicroscopy. Porcine cardiocytes (n = 75) were studied at baseline and in the presence of 80 micrograms/mL protamine. In addition, myocyte function was examined sequentially, first during treatment with 8 IU/mL heparin and then after the addition of a protamine dose sufficient to completely bind the heparin. The binding of heparin and protamine resulted in the formation of a heparin-protamine complex. The protamine concentration of 80 micrograms/mL is approximately equal to the serum concentration of protamine obtained in patients when administered in a dose of 5 mg/kg. In the presence of 80 micrograms/mL protamine, both percent shortening and velocity of shortening fell by more than 32% from baseline values (p < 0.05). The presence of either heparin alone or the heparin-protamine complex resulted in no change in baseline myocyte contractile measurements. Furthermore, to examine the effect of protamine on myocyte beta-adrenergic responsiveness a second series of experiments were performed. Myocyte contractile function was measured when 25 nmol/L isoproterenol was added to each of the protocols above. The presence of 80 micrograms/mL protamine resulted in a significant blunting of myocyte beta-adrenergic responsiveness. The presence of either heparin alone or the heparin-protamine complex resulted in no change in myocyte beta-adrenergic responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinesin is associated with a nonmicrotubule component of sea urchin mitotic spindles.

Sea urchin embryos in second division have been lysed into microtubule-stabilizing buffers to yield mitotic cytoskeletons (MCSs) that consist of two mitotic spindles surrounded by a cortical array of filaments. Microtubules have been completely extracted from MCSs by incubation at 0 degrees C with Ca2+-containing buffer. An antibody to the microtubule translocator kinesin stains the spindles in MCSs and in MCSs treated with 5 mM ATP and also stains spindle-remnants of the MCSs after the microtubules have been extracted. We conclude that kinesin binds to a nonmicrotubule component in the mitotic spindle. Based on these results, we present several models of kinesin function in the spindle.