Leona J. Rubin

Reduced L-type calcium current in ventricular myocytes from endotoxemic guinea pigs

The circulatory response to gram-negative sepsis and its experimental counterpart, endotoxemia, includes a profound dysfunction in myocardial contractility that is resident to the myocyte and associated with reduced systolic free intracellular Ca2+ concentration ([Ca2+]i). We explored the possibility that decreased systolic [Ca2+]iin endotoxemic myocytes is correlated with reduced L-type Ca2+ current ( I Ca,L). Ventricular myocytes were isolated from guinea pigs 4 h after an intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS; 4 mg/kg). Membrane potentials and Ca2+ currents were measured using whole cell patch-clamp methods. The action potential duration of endotoxemic myocytes was significantly shorter than control values (time to 50% repolarization: LPS, 314 ± 23 ms; control, 519 ± 36 ms, P < 0.05). Correspondingly, endotoxemic myocytes demonstrated significantly reduced peak I Ca,L density (3.5 ± 0.2 pA/pF) and Ba2+current ( I Ba) density (7.3 ± 0.5 pA/pF) compared with respective values of control myocytes ( I Ca,L density 6.1 ± 0.3 pA/pF, I Ba density 11.3 ± 0.8 pA/pF; P < 0.05). Endotoxemia-induced reduction in peak I Ca,L could not be attributed to alterations in current-voltage relationships, steady-state activation and inactivation, or recovery from inactivation. The β-adrenoceptor agonist isoproterenol, but not the Ca2+ channel activator BAY K 8644, reversed the LPS-induced reduction in peak I Ca,L, cell contraction, and systolic [Ca2+]i. These data demonstrate that part of the host response to endotoxemia involves diminished sarcolemmal I Ca,L of ventricular myocytes.The circulatory response to gram-negative sepsis and its experimental counterpart, endotoxemia, includes a profound dysfunction in myocardial contractility that is resident to the myocyte and associated with reduced systolic free intracellular Ca2+ concentration ([Ca2+]i). We explored the possibility that decreased systolic [Ca2+]i in endotoxemic myocytes is correlated with reduced L-type Ca2+ current (ICa,L). Ventricular myocytes were isolated from guinea pigs 4 h after an intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS; 4 mg/kg). Membrane potentials and Ca2+ currents were measured using whole cell patch-clamp methods. The action potential duration of endotoxemic myocytes was significantly shorter than control values (time to 50% repolarization: LPS, 314 +/- 23 ms; control, 519 +/- 36 ms, P < 0.05). Correspondingly, endotoxemic myocytes demonstrated significantly reduced peak ICa,L density (3.5 +/- 0.2 pA/pF) and Ba2+ current (IBa) density (7.3 +/- 0.5 pA/pF) compared with respective values of control myocytes (ICa,L) density 6.1 +/- 0.3 pA/pF, IBa density 11.3 +/- 0.8 pA/pF; P < 0.05). Endotoxemia-induced reduction in peak ICa,L could not be attributed to alterations in current-voltage relationships, steady-state activation and inactivation, or recovery from inactivation. The beta-adrenoceptor agonist isoproterenol, but not the Ca2+ channel activator BAY K 8644, reversed the LPS-induced reduction in peak ICa,L, cell contraction, and systolic [Ca2+]i. These data demonstrate that part of the host response to endotoxemia involves diminished sarcolemmal ICa,L of ventricular myocytes.

Endotoxin-induced myocardial dysfunction: is there a role for nitric oxide?

Nitric oxide has been implicated in the regulation of cardiac contractile function as well as the depressed myocardial contractility associated with sepsis and endotoxemia. We examined the effects of nitric oxide synthase (NOS) inhibitors and a nitric oxide generator on contractile responses of left atrial preparations and ventricular myocytes isolated from endotoxemic guinea pigs, which exhibit depressed myocardial contractile function. The NOS inhibitor L-NAME had no effect on contractile tension developed by control atria or atria isolated from guinea pigs 4 or 16 h after an intraperitoneal injection of endotoxin. Similarly, contraction of ventricular myocytes isolated from control or endotoxemic guinea pigs (4 h after endotoxin injection) was unchanged by exposure to several NOS inhibitors. In addition, neither Ca(2+)-dependent nor Ca(2+)-independent ventricular NOS activity was affected by endotoxemia. These data suggest that nitric oxide alone is not responsible for the cardiac contractile dysfunction of endotoxemic guinea pigs.

Differential Coronary Microvascular Exchange Responses to Adenosine: Roles of Receptor and Microvessel Subtypes

Objective: To assess the role of adenosine receptors in the regulation of coronary microvascular permeability to porcine serum albumin (PsPSA).

Contractile dysfunction of ventricular myocytes isolated from endotoxemic guinea pigs.

Mechanisms responsible for the decline in cardiac function following sepsis or endotoxemia are unclear but may result from indirect effects of cardiodynamic readjustments to diminishing venous return or to direct effects of endogenous factors on myocardial function. We examined contractile properties of ventricular myocytes isolated from endotoxemic guinea pig hearts to 1) verify and characterize inotropic dysfunction in the absence of immediate influences from extrinsic neurohumoral agents, and 2) assess the ability of β-adrenergic receptor activation to modulate contractility. Myocytes were isolated by enzymatic dispersion from hearts 4 h following an intraperitoneal injection of Escherichia coli endotoxin. Contractility was assessed using a computer-driven image analysis system. Inotropic responsiveness of endotoxemic myocytes to changes in frequency of stimulation (.2–2.0 Hz) or increases in extracellular calcium ([Ca2+]o, 1.8–8.0 mM) was significantly less than control myocytes, even with maximally effective frequencies or [Ca2+]o. These data demonstrate that the endotoxin-induced dysfunction is intrinsic to ex vivo cardiac myocytes and independent of immediate influence from extracardiac factors by 4 h in vivo exposure to endotoxemia. Inotropic responsiveness to β-adrenergic receptor activation remained intact in endotoxemic myocytes; maximally effective concentrations (>10 nM) reversed the endotoxin-induced contractile dysfunction. These data confirm that E. coli endotoxemia incorporates intrinsic contractile dysfunction of myocardial cells, while sparing their ability to respond to inotropic mechanisms activated by β-adrenoceptor agonists.

Isoform-selective 5'-AMP-activated protein kinase-dependent preconditioning mechanisms to prevent postischemic leukocyte-endothelial cell adhesive interactions

We previously demonstrated that preconditioning induced by ethanol consumption at low levels [ethanol preconditioning (EPC)] or with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR-PC) 24 h before ischemia-reperfusion prevents postischemic leukocyte-endothelial cell adhesive interactions (LEI) by a mechanism that is initiated by nitric oxide formed by endothelial nitric oxide synthase. Recent work indicates that 1) ethanol increases the activity of AMP-activated protein kinase (AMPK) and 2) AMPK phosphorylates endothelial nitric oxide synthase at the same activation site seen following EPC (Ser1177). In light of these observations, we postulated that the heterotrimeric serine/threonine kinase, AMPK, may play a role in triggering the development of the anti-inflammatory phenotype induced by EPC. Ethanol was administered to C57BL/6J mice by gavage in the presence or absence of AMPK inhibition. Twenty-four hours later, the numbers of rolling and adherent leukocytes in postcapillary venules of the small intestine were recorded using an intravital microscopic approach. Following 45 min of ischemia, LEI were recorded after 30 and 60 min of reperfusion or at equivalent time points in control animals. Ischemia-reperfusion induced a marked increase in LEI relative to sham-operated control mice. The increase in LEI was prevented by EPC, an effect that was lost with AMPK inhibition during the period of ethanol exposure. Studies conducted in AMPK α(1)- and α(2)-knockout mice suggest that the anti-inflammatory effects of AICAR are not dependent on which isoform of the catalytic α-subunit is present because a deficiency of either isoform results in a loss of protection. In sharp contrast, EPC appears to be triggered by an AMPK α(2)-isoform-dependent mechanism.

Endotoxemia-induced myocardial dysfunction is not associated with changes in myofilament Ca2+ responsiveness

Myocardial contractile function is depressed after onset of endotoxemia and is intrinsic to the ventricular myocyte. We tested the hypothesis that decreased Ca2+ responsiveness of the contractile myofilaments underlies this inotropic depression. Specifically, we evaluated the relationship between Ca2+ and unloaded cell shortening and isometric tension development of skinned guinea pig ventricular myocytes. Myocytes were isolated 4 h after intraperitoneal injection of 4 mg/kg Escherichia colilipopolysaccharide (LPS) or saline (control; Ctl). Myofilament Ca2+ responsiveness assessed by image analysis of shortening of skinned myocytes at pH 7.0 was not different between Ctl [pCa value that resulted in half-maximal shortening (pCa50): 5.78 ± 0.04] and LPS (pCa50: 5.72 ± 0.02). Similarly, myofilament Ca2+ responsiveness measured by isometric tension of skinned myocytes was not different between Ctl (pCa50: 5.73 ± 0.02) and LPS (pCa50: 5.76 ± 0.02). Maximal tension generated by LPS myocytes (2.89 ± 0.23 g/mm2) was significantly less ( P < 0.05) than Ctl (3.75 ± 0.34 g/mm2). However, when myocytes were isolated and skinned in the presence of protease inhibitors, maximal tension generated by LPS myocytes (3.53 ± 0.98 g/mm2) was similar to Ctl (3.01 ± 0.80 g/mm2). We conclude that in vivo administration of LPS resulting in endotoxemia without shock does not alter myofilament Ca2+ responsiveness of ventricular myocytes. Rather, reduced contractility is more likely a result of decreased Ca2+ availability because systolic Ca2+ transients of fura 2-loaded LPS myocytes were significantly decreased ( P < 0.05) compared with Ctl myocytes.

Cardiodynamic response to Escherichia coli endotoxemia: effects of fluid resuscitation.

We tested the influence of in vivo volume resuscitation on intrinsic contractile properties of left ventricular (LV) preparations of endotoxemic guinea pigs. Escherichia coli endotoxin (LPS)-injected animals were divided into nonresuscitated and resuscitated groups. Volume resuscitation improved cardiac output and stroke volume, increased arterial pH and body temperature, and decreased mortality. In isovolumetric LV preparations isolated 4 h after LPS injection, LV systolic pressures (in mmHg) of LPS with (42 ± 3) and without (42 ± 2) fluid resuscitation were consistently less than control values (70 ± 3). LV end-diastolic pressure-volume (compliance) decreased in LPS-nonresuscitated hearts, while LV compliance of LPS-resuscitated hearts was similar to control. Thus, intravascular volume expansion selectively improved LV diastolic compliance of LPS hearts without affecting LV systolic function. These findings suggest that LV systolic and diastolic dysfunctions associated with endotoxemia and Gram-negative sepsis may involve separate pathogenic mechanisms.

Pulsed Estrogen Therapy Prevents Post-OVX Porcine Dura Mater Microvascular Network Weakening via a PDGF-BB-Dependent Mechanism

In postmenopausal women, estrogen (E2) deficiencies are frequently associated with higher risk of intracranial hemorrhage, increased incidence of stroke, cerebral aneurysm, and decline in cognitive abilities. In younger postpartum women and those using oral contraceptives, perturbations in E2 are associated with higher risk of cerebral venous thrombosis. A number of serious intracranial pathologic conditions linked to E2 deficiencies, such as dural sinus thrombosis, dural fistulae, non-parenchymal intracranial hemorrhages, migraines, and spontaneous cerebrospinal fluid leaks, involve the vessels not of the brain itself, but of the outer fibrous membrane of the brain, the dura mater (DM). The pathogenesis of these disorders remains mysterious and how estrogen regulates structural and functional integrity of DM vasculature is largely unknown. Here, we demonstrate that post ovariectomy (OVX) DM vascular remodeling is manifested by microvessel destabilization, capillary rarefaction, increased vascular permeability, and aberrant angio-architecture, and is the result of disrupted E2-regulated PDGF-BB signaling within dura microvasculature. These changes, associated with the reduction in systemic PDGF-BB levels, are not corrected by a flat-dose E2 hormone replacement therapy (HRT), but are largely prevented using HRT schedules mimicking physiological E2 fluctuations. We demonstrate that 1) E2 regulates PDGF-BB production by endothelial cells in a dose-dependent manner and 2) optimization of PDGF-BB levels and induction of robust PDGF-mediated endothelial cell-vascular pericyte interactions require high (estrous) E2 concentrations. We conclude that high (estrous) levels of E2 are important in controlling PDGF-mediated crosstalk between endothelial cells and pericytes, a fundamental mechanism governing microvessel stability and essential for preserving intracranial homeostasis.

Dietary genistein induces sex-dependent effects on murine body weight, serum profiles, and vascular function of thoracic aortae.

BACKGROUND The influence on, or interaction of, sex and dietary genistein on serum markers of cardiovascular health and cardiovascular function remain unclear. OBJECTIVES Our purpose was to examine the effects of a genistein-containing diet (600 mg/kg food) (600G) and a genistein-free diet (0G), on cardiovascular risk parameters of male and female mice. METHODS C57BL/6J mice were fed the diets for 1 month, after which time blood pressure, serum markers, and in vitro vascular reactivity was measured. RESULTS Males fed the 600G diet gained significantly less weight than males fed the 0G diet (by 1.71 g); diet had no effect on female weight gain. Males fed the 600G diet also exhibited significantly elevated serum insulin (2.9 [0.5] vs 1.8 [0.4] ng/dL), and decreased serum glucose (0.15 [0.01] vs 0.24 [0.02] ng/dL) levels, resulting in a significant increase in the ratio of insulin to glucose; insulin and glucose levels were not changed by dietary genistein in females. Arterial pressure measurements from 0G-fed males were lower than other groups. However, basal vascular reactivity of isolated aortic rings was significantly increased by the 600G diet in both males (from 0.55 [0.03] to 0.94 [0.18] g) and females (from 0.45 [0.04] to 0.78 [0.09] g). Aortic wall thickness was not affected by diet. Norepinephrine-mediated contractility was also greater in aortic rings of male and female mice fed the 600G diet, and differences from the 0G diet persisted in the presence of L-NG-nitroarginine methyl ester but were completely accounted for by increased basal reactivity. CONCLUSION Our data indicate that 1 month of a 600G or 0G diet significantly alters vascular function independent of sex. In contrast, sex-dependent differences exist in well-established serum markers of cardiovascular health and disease.

Cytosolic Ca2+ concentration and contraction-relaxation properties of ventricular myocytes from Escherichia coli endotoxemic guinea pigs: effect of fluid resuscitation.

Hearts isolated from a guinea pig model of Escherichia coli endotoxemia exhibit decreased systolic contractile function and reduced diastolic compliance of the left ventricle within 4 h after injection of endotoxin. Fluid resuscitation prevented the endotoxin-induced decrease in diastolic compliance without affecting systolic contractile depression. Because intrinsic myocardial dysfunction after endotoxemia may result from alterations in intracellular Ca2+ handling, we tested the hypothesis that in vivo fluid resuscitation improved diastolic function by altering Ca2+ handling of the myocardium. We tested this hypothesis by measuring cell shortening and intracellular Ca2+ of ventricular myocytes isolated from endotoxemic guinea pigs. E. coli endotoxin (LPS, 1 mg/kg)-injected guinea pigs were divided into resuscitated and nonresuscitated groups. Fluid resuscitated animals received a Ringers infusion (8 mL·kg−1·h−1) intravenously (i. v.) beginning immediately after endotoxin injection. Four hours later, ventricular myocytes were isolated enzymatically and loaded with fura-2/AM. When myocytes were field stimulated at .8 Hz, peak systolic Ca2+ transients of LPS-resuscitated (619 ± 75 nM) and LPS-nonresuscitated (599 ± 60 nM) myocytes were not significantly different from each other, but both were significantly less than values from control myocytes (1187 ± 118 nM, p <.05). The percentage of cell shortening of LPS-resuscitated (6.2 ±.9%) and LPS-nonresuscitated (6.2 ±.3%) myocytes were also less than control (11.8 ±.5%, p < .05). In contrast to improved diastolic compliance of fluid-resuscitated hearts, diastolic [Ca2+], of myocytes (at .8 Hz) from LPS-resuscitated animals (138 ± 47 nM) was not statistically different from LPS-nonresuscitated animals (129 ± 19 nM). Diastolic values of both LPS groups were consistently lower than control value (251 ± 38 nM, p <.05). These data suggest that improved diastolic compliance of LPS hearts following fluid resuscitation is not associated with improved myocyte contractility or myoplasmic Ca2+ handling.