William R. Law

Breakdown and Release of Myofilament Proteins During Ischemia and Ischemia/Reperfusion in Rat Hearts Identification of Degradation Products and Effects on the pCa-Force Relation

Our objective in experiments reported here was to identify myofilament proteins of rat hearts either lost or degraded by cardiac ischemia (15- or 60-minute duration) with and without 45 minutes of reperfusion. We correlated these changes with alterations in myofilament sensitivity to Ca2+ and maximum force generation. Protein degradation and loss were assessed by high-performance liquid chromatography, SDS-PAGE, Western blotting analysis, and amino acid sequencing. Compared with nonischemic control hearts, bundles of skinned fibers from hearts subjected to ischemia alone demonstrated a decrease in maximum force generation and an increase in sensitivity to Ca2+. These changes in function were increased with the duration of the ischemia and with reperfusion. With increasing duration of ischemia, there was an increased loss and degradation of myofibrillar alpha-actinin and troponin I (TnI) at its C-terminus. Alpha-actinin and TnI were most susceptible to ischemia, but with 60 minutes of ischemia/reperfusion, there was also degradation of myosin light chain-1 (MLC1) involving a clip of residues 1 to 19. The MLC1 degradation product was detected in the reperfusion effluent (along with troponin T, tropomyosin, and alpha-actinin) but not in the tissue with 60 minutes of ischemia with no reperfusion. Moreover, with ischemia the following proteins became associated with the myofibrils: GAPDH and proteins of the mitochondrial ATP synthase complex. Our results provide new evidence regarding the mechanism by which ischemia/reperfusion causes myocardial injury and support the hypothesis that an important element in the injury is altered activity and structure of the myofilaments.

The Effects Of Diaspirin Cross-linked Hemoglobin In Sepsis

We tested the hypothesis that diaspirin cross-linked hemoglobin (DCLHb; Baxter Healthcare Corp.) would improve blood pressure, organ perfusion, and mortality during sepsis. Rats were catheterized to assess general hemodynamics (protocol 1) or regional blood flow (protocol 2). Sepsis was induced by intraperitoneal introduction of a cecal slurry (100 mg/kg). In protocol 1, rats received either 100 or 250 mg/kg DCLHb, or albumin at 1, 2, or 4 h after sepsis induction. Hemodynamics were recorded at these times and daily for 72 h. DCLHb increased blood pressure, prevented 72 h leukocytosis, and reduced mortality, but the timing of DCLHb administration was crucial. In protocol 2 only moribund septic animals received 100 mg/kg DCLHb or iso-oncotic albumin i.v. Hemodynamics and regional organ blood flows were measured at baseline, immediately before and after treatment, and at 24 h. DCLHb immediately increased blood pressure with no changes in cardiac output, heart rate, or regional perfusion. DCLHb increased regional perfusion to vital areas at 24 h (compared to albumin group). Distribution of cardiac output in albumin-treated rats was significantly skewed toward skeletal muscle at a time when cardiac output was significantly lower as compared with DCLHb treated animals. In conclusion, DCLHb safely elicited a pressor response, and improved regional perfusion to selected tissues. However, DCLHb benefits were best obtained when given within a specific time frame.

Effect of NG-nitro-L-arginine methyl ester on testicular blood flow and serum steroid hormones during sepsis.

ABSTRACT Production of nitric oxide (NO) via NO synthase (NOS) has been implicated in the regulation of steroidogenesis in normal physiology and septic pathophysiology. The hypothesis that blockade of NOS by NG-nitro-L-arginine methyl ester (L-NAME) would affect testicular blood flow and circulating levels of steroid reproductive hormones was tested. Male Sprague-Dawley rats (350–450 g) were randomized to septic and nonseptic groups. Sepsis was induced with an intraperitoneal (i.p.) injection of a cecal slurry (200 mg/kg in 5 mL 5% dextrose in water (D5W)) in rats, while nonseptic rats received only sterile D5W. The rats (n = 6 per group) were catheterized in the jugular vein, left ventricle (via right carotid artery), and tail artery to determine blood flow and systemic hemodynamics and to collect blood at 24 h after induction of sepsis/sham sepsis. After baseline (24 h post-cecal slurry challenge) measurement, L-NAME (.50 mg/ kg-min) was infused through the jugular vein for 10 min, blood flow was determined using a radioactive microsphere technique, and blood samples were collected. The serum concentrations of corticosterone, progesterone, and testosterone were determined using radioimmunoassay. Plasma concentrations of NO byproducts (NOx) were determined using the Greiss reaction. After 24 h, heart rate, testicular blood flow, and NOx levels were significantly increased, whereas the serum concentration of testosterone was significantly decreased in the septic group as compared with the nonseptic group. However, serum concentrations of progesterone and corticosterone at 24 h after induction of sham-sepsis or sepsis were not statistically different. Infusion of L-NAME significantly reduced the testicular blood flow and serum NOx levels in septic rats as compared with their baseline values. The administration of L-NAME significantly increased the concentration of testosterone in nonseptic and septic rats as compared with their respective basal values. However, testosterone levels in septic rats were still significantly lower than in nonseptic rats. The results of this study indicate that the synthesis of NO through NO synthase may play a role in the regulation of testicular blood flow and the serum levels of testosterone, associated with chronic peritoneal sepsis in the rat.

Adenosine Reversal of In Vivo Hepatic Responsiveness to Insulin

Modulation by adenosine of hepatic responsiveness to insulin was investigated in vivo in 10 healthy mongrel dogs of both sexes by determining net hepatic glucose output (NHGO) in response to insulin during the presence or absence of exogenous adenosine infusion. In addition, two separate series of experiments were performed to study the effect of adenosine (n = 7) or glucagon (n = 5) on NHGO. Basal NHGO, quantitated via the Fick principle, was significantly decreased by insulin infusion (4 U/min; 4.8 ± 0.6 vs. −1.7 ± 2.6 mg · kg−1 · min−1, P < 0.05). The addition of an intrahepatic arterial infusion of adenosine (10 μmol/min) during insulin infusion caused glucose output to return to basal levels (insulin, −1.7 ± 2.6 mg · kg−1 · min−1; insulin + adenosine, 3.8 ± 1.6 mg · kg−1 · min−1, P < 0.05). The addition of intrahepatic arterial saline (control) during insulin infusion had no effect on insulins action (insulin, −1.0 ± 1.9 mg · kg−1 · min−1; insulin + saline, −1.2 ± 1.6 mg · kg−1 · min−1, P > 0.05). Hepatic glucose, lactate, and oxygen deliveries were not affected during either insulin or insulin plus adenosine infusion. Intrahepatic arterial infusion of adenosine alone had no effect on NHGO, whereas intrahepatic arterial infusion of glucagon alone stimulated glucose output approximately fivefold (basal, 2.7 ± 0.4 mg · kg−1 · min−1; glucagon, 15.5 ± 1.2 mg · kg−1 · min−1, P < 0.01). These results show that adenosine completely reversed the inhibition by insulin of NHGO. These data suggest that adenosine may act as a modulator of insulin action on the liver.

Therapeutic potential for transient inhibition of adenosine deaminase in systemic inflammatory response syndrome

ObjectiveWe sought to determine the potential usefulness of 2′-deoxycoformycin (pentostatin), an inhibitor of adenosine deaminase, as a postinsult, or prophylactic treatment for systemic inflammatory response syndrome resulting from fecal peritonitis. DesignProspective, randomized, controlled experiment. SettingSmall animal basic science laboratory. SubjectsMale Spague-Dawley rats, weighing 300 to 350 g. InterventionsRats with fecal peritonitis (intraperitoneal cecal slurry) were treated with 1 mg/kg pentostatin intraperitoneally 24 hrs before, or intravenously when signs of illness presented (2 hrs after induction of peritonitis). Signs of illness included tachycardia, tachypnea, and leukopenia. All rats received 50 mL/kg 0.9% saline resuscitative fluid at 2 hrs. Measurements and Main ResultsSurvival to day 6 was 100% in nonseptic sham rats, but 33% in untreated septic rats. In rats given pentostatin either 2 hrs after the insult, or 24 hrs before the insult, 6-day survival improved to 81% and 78%, respectively. Histology revealed diffuse peritonitis, and evidence of systemic inflammatory response syndrome, including local and distant site vascular damage and leukocyte activation. These responses to the septic challenge were abrogated by pentostatin treatment. Return of significant amount of tissue adenosine deaminase activity by 24 hrs and later recovery of white blood cell counts argue against any potential for inappropriate immunosuppression by pentostatin. ConclusionsThese data indicate that the novel use of pentostatin to prevent systemic inflammatory response syndrome secondary to fecal peritonitis shows uncommon promise as a therapeutic tool. All indices of systemic inflammatory response syndrome were abrogated and survival improved when pentostatin was not given until after signs of the illness became manifest. Because protection was afforded with treatment 24 hrs in advance of the inciting insult, pentostatin also has the unique potential for use as a true prophylactic agent.

Splanchnic vascular control during sepsis and endotoxemia.

Endotoxemia and sepsis often result in circulatory derangements which manifest as perfusion maldistributions. It has been widely accepted that the splanchnic circulation decreases in perfusion during advanced septic or endotoxemic states. Impaired perfusion of splanchnic organs may result not only in organ dysfunction but also exacerbations of polymicrobial bacteremia due to intestinal mucosal leakage. Consequently, evaluation of the splanchnic mechanisms of vasoregulation and how perfusion is maintained is vital to any topic concerning the management of the septic patient.

Starling resistor effects on pulmonary artery occlusion pressure in endotoxin shock provide inaccuracies in left ventricular compliance assessments.

OBJECTIVESnAssessment of left ventricular preload and left ventricular compliance changes in septic shock using pulmonary artery occlusion pressure (PAOP) presumes that this pressure accurately reflects left heart filling pressure. We tested the hypothesis that Starling resistor forces render PAOP inaccurate as an index of left heart filling pressure, resulting in misleading assessments of left ventricular compliance changes.nnnDESIGNnProspective, randomized, controlled study.nnnSETTINGnLarge-animal research laboratory at a university.nnnSUBJECTSnFourteen anesthetized domestic pigs weighing 20 to 25 kg.nnnINTERVENTIONSnPulmonary artery flotation catheters and systemic arterial catheters were placed via right cervical vessels. The left atrium was directly catheterized for left atrial pressure measurements. Left ventricular end-diastolic diameter was measured using sonomicrometry. Other measured or calculated variables were mean arterial pressure, mean pulmonary arterial pressure, PAOP, pulmonary capillary pressure, and pulmonary arterial and venous resistances. Pigs received endotoxin (0.5 mg/kg i.v. over 30 mins), or an equivalent volume of saline. At t = 60 mins, pigs were resuscitated with lactated Ringers solution (40 mL/kg over 30 mins). Measurements were taken before and after endotoxin administration, and immediately and 30 mins after lactated Ringers solution administration. Data were analyzed by two-way analysis of variance (p < or = .05).nnnMEASUREMENTS AND MAIN RESULTSnPAOP, mean pulmonary arterial pressure, and pulmonary capillary pressure increased after endotoxin infusion, while left atrial pressure and left ventricular end-diastolic diameter decreased. Left atrial pressure and left ventricular end-diastolic diameter returned to baseline immediately after lactated Ringers solution administration, while PAOP remained increased. Pulmonary arterial resistance and pulmonary venous resistance increased after endotoxin administration, with pulmonary venous resistance showing the greater percent increase. Pulmonary venous resistance decreased transiently immediately after lactated Ringers solution administration. These changes were not observed in the control group. Accordingly, comparisons of PAOP vs. left ventricular end-diastolic diameter, and left atrial pressure vs. left ventricular end-diastolic diameter yielded divergent results.nnnCONCLUSIONSnThe dissociation between PAOP and left atrial pressure, while left ventricular and -diastolic diameter (preload volume) decreased, and changes in pulmonary venous resistance, are strong evidence for Starling resistor forces (venocompression) rather than active venoconstriction. These data indicate that PAOP overestimates left atrial pressure during endotoxin shock, making it an inaccurate index of left ventricular preload. This overestimation can cause misleading assessments of left ventricular compliance.

Porcine peritoneal sepsis: modeling for clinical relevance.

The characteristics of two types of intraperitoneal (i.p.) soilage sepsis models, autologous fecal inoculum (FEC) and a pure culture of Escherichia coli (EC), were studied in 26 male Yucatan minipigs (20–30 kg). Early (1–4 h) and late (24–72 h) changes were different between the two groups. The EC group was characterized early by hypotension, low cardiac output, and increased systemic and pulmonary vascular resistances, along with leukopenia, hypoglycemia, lactacidemia, and elevated blood urea nitrogen. Of the pigs in the EC group that survived the early effects, there were few significant differences in physiological parameters, compared to control pigs, that would indicate ongoing pathological processes. In contrast, the FEC group pigs demonstrated early hypotension, but with increased cardiac output and reduced systemic vascular resistance. Other parameter changes were similar to those seen in the EC pigs, but to a lesser degree, with the exception of elevations in serum lactate dehydrogenase. Also in contrast to the EC group, most of the changes in the FEC group persisted in later days, and FEC pigs demonstrated leukocytosis. There were also greater elevations in circulating lipopolysaccharide (LPS) concentrations in the EC group that returned later to baseline levels. In the FEC group, there were persistently elevated LPS concentrations over 72 h. These observations suggest that pigs challenged with intraperitoneal E. coli demonstrated an initial acute peritonitis and damaging physiologic effects of high levels of circulating LPS. Survivors in this group improved and were physiologically stable after 24 h. Pigs that received i.p. autologous feces developed an early acute peritonitis phase with lower levels of circulating LPS, and later developed pronounced peritoneal reaction as demonstrated by multiple abdominal abscesses, pyogenic granuloma formation, and adhesions with physiological evidence of developing sepsis over 72 h. These observations indicate that i.p. EC models evoke a systemic response not unlike intravenous administration of LPS or EC, however, the FEC model produced a systemic response akin to a slower developing septic process.

Elevated coronary endothelin-1 but not Nitric oxide in diabetics during CABG

BACKGROUNDnAfter coronary artery bypass grafting procedures, a higher incidence of morbidity and mortality has been reported in diabetic patients. We tested whether coronary artery bypass grafting in diabetics affects the endothelin-1 and nitric oxide coronary effluent profile during reperfusion.nnnMETHODSnTwenty-one consecutive patients (9 with type II diabetes mellitus, 12 non-diabetics) underwent coronary artery bypass grafting by one surgeon. The two groups did not differ in preoperative ejection fraction, Parsonnet score, number of vessels bypassed, or cross-clamp time. Each patient was treated in the same intraoperative manner with single atrial, aortic, and antegrade and retrograde cardioplegia (CPL) cannulas. Cold CPL arrest was by antegrade and retrograde infusion of modified Buckberg CPL solution. Warm CPL solution was infused before reperfusion. Coronary sinus blood samples were obtained for estimation of endothelin-1 and nitrite plus nitrate before CPL arrest and at 1 and 15 minutes after each of 2 reperfusion periods.nnnRESULTSnIn diabetics, endothelin-1 was significantly increased at all reperfusion times as compared with non-diabetics. Nitrite plus nitrate levels were significantly higher in patients with diabetes than in those without, but did not change with time in either of the groups.nnnCONCLUSIONSnReperfusion after CPL during coronary artery bypass grafting procedure can trigger the release of endothelin-1 in patients with diabetes mellitus. This may favor increased vascular tone or positive inotropic responses after coronary artery bypass grafting and may contribute to significant cardiovascular consequences in diabetic patients.

Myocardial insulin resistance during acute endotoxin shock in dogs.

Myocardial insulin responsiveness was determined in open-chest pentobarbital sodium-anesthetized dogs before and after endotoxin administration. Animals were instrumented to measure mean arterial blood pressure (MABP), heart rate (HR), and coronary blood flow. Myocardial glucose uptake and myocardial oxygen uptake (MVO2) were determined during a basal control period and after a hyperinsulinemic-euglycemic clamp procedure over a wide range of insulin concentrations. The clamp was accomplished by intravenously infusing insulin (0–4000 mU/min) and 20% glucose in sufficient amounts to maintain arterialglucose concentrations within 5 mg/dl of the control value. In a separate series of experiments, myocardial insulin responsiveness was determined by use of a single dose of insulin (4000 mU/min). This was done to determine whether antecedent insulin infusions during the sequential clamp procedure would affect the responsiveness of the heart. In control experiments, myocardial glucose uptake increased without any changes in HR, MVO2, or MABP. Maximum myocardial glucose uptake occurred at an insulin infusion rate between 400 and 4000 mU/min. A single concentration of insulin resulted in similar increases in myocardial glucose uptake as with the sequential clamp protocol. Acute endotoxin shock was induced by bolus injection of 1 mg/kg Salmonella typhimurium endotoxin (Difco Labs, Detroit, MI). One hour after administration of endotoxin, basal myocardial glucose uptake was decreased compared with the control animals. After 1 h of endotoxin shock, the heart was refractory to all concentrations of insulin, suggesting the site for altered insulin response was being mediated by a postreceptor mechanism.