Stephen M. Richards

Improved Preservation of Saphenous Vein Grafts by the Use of Glyceryl Trinitrate–Verapamil Solution During Harvesting

BACKGROUND High-pressure distension during harvesting damages the saphenous vein (SV) and may contribute to subsequent coronary artery bypass graft (CABG) occlusion. Application of vasodilator agents to the SV during harvesting may reduce the need for high-pressure distension and improve graft quality. We tested the effects of a vasodilator solution containing glyceryl trinitrate and verapamil (GV) or the conventional agent papaverine (Pap) on the pressure necessary to overcome SV spasm and on the structure and biochemistry of the SV graft. METHODS AND RESULTS Thirty-six patients undergoing CABG were randomly allocated to receive an application of either topical and intraluminal GV solution, topical Pap, or topical and intraluminal Ringers solution (untreated) to the SV during harvesting. The peak and mean pressures required to distend the vein were recorded. Samples of SV were taken for microscopy and biochemical analysis just before we performed the anastomosis. The percentage of endothelial coverage was calculated by area measurements of stained en face preparations of the vein intima. The results for peak pressures (mmHg) were: untreated, 479.2 +/- 27.5; Pap, 384.8 +/- 29.0; and GV, 309.5 +/- 28.3 (P < .001, GV plus Pap versus untreated); and the results for mean pressures (mm Hg) were untreated, 136.2 +/- 9.6; Pap, 102.2 +/- 10.8; and GV, 98.0 +/- 8.3 (P < .01, GV plus Pap versus untreated). The results for endothelial cover (%) were: untreated, 43.7 +/- 7.0; Pap, 44.1 +/- 9.2; and GV, 68.7 +/- 7.0 (P < .05, GV versus Pap); and the results for ATP (nmol/g wet wt) were: untreated, 67.3 +/- 12.7; Pap, 112.0 +/- 19.4; and GV, 132.5 +/- 22.7 (P < .05, GV plus Pap versus untreated). CONCLUSIONS First, pharmacological treatment of SV during harvesting, especially with GV solution, allows the use of a lower distension pressure and reduces the breakdown of high-energy phosphates in the vein wall. Second, topical and intraluminal use of GV solution during vein harvesting improves endothelial coverage compared with the topical use of Pap or no pharmacological treatment.

Decreased microvascular vasomotion and myogenic response in rat skeletal muscle in association with acute insulin resistance

In addition to increased glucose uptake, insulin action is associated with increased total and microvascular blood flow, and vasomotion in skeletal muscle. The aim of this study was to determine the effect of acute insulin resistance caused by the peripheral vasoconstrictor α‐methylserotonin (αMT) on microvascular vasomotion in muscle. Heart rate (HR), mean arterial pressure (MAP), femoral blood flow (FBF), whole body glucose infusion (GIR) and hindleg glucose uptake (HGU) were determined during control and hyperinsulinaemic euglycaemic clamp conditions in anaesthetized rats receiving αMT infusion. Changes in muscle microvascular perfusion were measured by laser Doppler flowmetry (LDF) and vasomotion was assessed by applying wavelet analysis to the LDF signal. Insulin increased GIR and HGU. Five frequency bands corresponding to cardiac, respiratory, myogenic, neurogenic and endothelial activities were detected in the LDF signal. Insulin infusion alone increased FBF (1.18 ± 0.10 to 1.78 ± 0.12 ml min–1, P < 0.05), LDF signal strength (by 16% compared to baseline) and the relative amplitude of the myogenic component of vasomotion (0.89 ± 0.09 to 1.18 ± 0.06, P < 0.05). When infused alone αMT decreased LDF signal strength and the myogenic component of vasomotion by 23% and 27% respectively compared to baseline, but did not affect HGU or FBF. Infusion of αMT during the insulin clamp decreased the stimulatory effects of insulin on GIR, HGU, FBF and LDF signal and blocked the myogenic component of vasomotion. These data suggest that insulin action to recruit microvascular flow may in part involve action on the vascular smooth muscle to increase vasomotion in skeletal muscle to thereby enhance perfusion and glucose uptake. These processes are impaired with this model of αMT‐induced acute insulin resistance.

Loss of insulin-mediated microvascular perfusion in skeletal muscle is associated with the development of insulin resistance

Aim: The aetiology of the development of type 2 diabetes remains unresolved. In the present study, we assessed whether an impairment of insulin‐mediated microvascular perfusion occurs early in the onset of insulin resistance.

Differing protection with aspartate and glutamate cardioplegia in the isolated rat heart

Aspartate and glutamate each have been shown to improve cardiac recovery after hypoxia or ischemia under normothermic conditions, but whether their effects are additive and to what extent they are modified by hypothermia has not been studied systematically. We set out to compare the individual and combined protective effects of aspartate and glutamate during cardioplegic arrest under normothermic and hypothermic conditions in the rat. Using isolated working rat hearts, functional and metabolic recovery was assessed after 0.5 hours of potassium arrest at 37 degrees C or 5 hours at 2 degrees C in control hearts (C) and in hearts in which 20 mmol/L glutamate (G), 20 mmol/L aspartate (A), or both (A + G) was added to the cardioplegic solution. Under normothermic conditions, percentage recovery of prearrest work (mean +/- standard error of the mean) was as follows: C = 31.7 +/- 2.8, G = 34.8 +/- 0.2, A = 49.6 +/- 2.8*, A + G = 53.7 +/- 2.3*. Under hypothermic conditions, the values were as follows: C = 40.4 +/- 4.0, G = 45.2 +/- 2.3, A = 59.4 +/- 1.8*, A + G = 54.1 +/- 1.2* (*p < 0.01 versus C and G). Recovery of postischemic high-energy phosphate content followed the same pattern: A = A + G > G or C. Measurement of postischemic myocardial content of amino acids showed that recovery of function and energy status correlated with maintenance of myocardial levels of aspartate (r = 0.9; p < 0.01) but not glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic and vascular actions of endothelin-1 are inhibited by insulin-mediated vasodilation in perfused rat hindlimb muscle.

1 Endothelin‐1 (ET‐1) is a potent endothelium‐derived vasoactive peptide and may be involved in the microvascular actions of insulin for the normal delivery of nutrients to muscle, although higher levels may be antagonistic. 2 Our aim was to observe the interaction between ET‐1 and insulin. Initially, we attempted to distinguish the vascular from the metabolic effects of ET‐1 in the constant‐flow pump‐perfused rat hindlimb by using various doses of ET‐1 and measuring changes in perfusion pressure (PP), oxygen consumption (VO2), glucose uptake (GU) and lactate release (LR). Sodium nitroprusside (SNP) was used to block vasoconstriction and to thus assess the relationship between vascular and metabolic effects. Insulin was included in later experiments to determine the interaction between insulin and ET‐1 on the above parameters. 3 ET‐1 caused a dose‐dependent increase in PP. Effects on VO2 were biphasic, with low doses increasing VO2, and higher doses leading to a net inhibition. GU and LR were increased at lower doses (ET‐1 1 nM), but this effect was lost at higher doses (10 nM ET‐1). 4 SNP (50 μM) fully blocked the increase in pressure and metabolism due to low‐dose ET‐1 and partly blocked both pressure and metabolic responses by the high dose. ET‐1 vasodilatory activity was minimal at high or low dose. Insulin (15 nM) alone caused GU, which was not affected by ET‐1. Of the other parameters measured, insulin behaved essentially the same as SNP, inhibiting the pressure and oxygen effects. 5 Overall, these results show that ET‐1 has a biphasic dose‐dependent vasoconstrictor effect on hindlimb blood vessels, able to modulate flow to cause both the stimulation and inhibition of metabolism, although these effects are blocked by insulin, which is able to vasodilate against both low and high doses of ET‐1.

Obesity, insulin resistance, and capillary recruitment

ABSTRACT

Adiponectin opposes endothelin-1-mediated vasoconstriction in the perfused rat hindlimb

Recent studies have shown that adiponectin is able to increase nitric oxide (NO) production by the endothelium and relax preconstricted isolated aortic rings, suggesting that adiponectin may act as a vasodilator. Endothelin-1 (ET-1) is a potent vasoconstrictor, elevated levels of which are associated with obesity, type 2 diabetes, hypertension, and cardiovascular disease. We hypothesized that adiponectin has NO-dependent vascular actions opposing the vasoconstrictor actions of ET-1. We studied the vascular and metabolic effects of a physiological concentration of adiponectin (6.5 μg/ml) on hooded Wistar rats in the constant-flow pump-perfused rat hindlimb. Adiponectin alone had no observable vascular activity; however, adiponectin pretreatment and coinfusion inhibited the increase in perfusion pressure and associated metabolic stimulation caused by low-dose (1 nM) ET-1. Adiponectin was not able to oppose vasoconstriction when infusion was commenced after ET-1. This is in contrast to the NO donor sodium nitroprusside, which significantly reduced the pressure due to established ET-1 vasoconstriction, suggesting dissociation of the actions of adiponectin and NO. In addition, adiponectin had no effect on vasoconstriction caused by either high-dose (20 nM) ET-1 or low-dose (50 nM) norepinephrine. Our findings suggest that adiponectin has specific, apparently NO-independent, vascular activity to oppose the vasoconstrictor effects of ET-1. The hemodynamic actions of adiponectin may be an important aspect of its insulin-sensitizing ability by regulating access of insulin and glucose to myocytes. Imbalance in the relationship between adiponectin and ET-1 in obesity may contribute to the development of insulin resistance and cardiovascular disease.

Muscle insulin resistance resulting from impaired microvascular insulin sensitivity in Sprague Dawley rats

AIMS Enhanced microvascular perfusion of skeletal muscle is important for nutrient exchange and contributes ∼40% insulin-mediated muscle glucose disposal. High fat-fed (36% fat wt./wt.) rats are a commonly used model of insulin-resistance that exhibit impairment of insulin-mediated microvascular recruitment and muscle glucose uptake, which is accompanied by myocyte insulin-resistance. Distinguishing the contribution of impaired microvascular recruitment and impaired insulin action in the myocyte to decreased muscle glucose uptake in these high-fat models is difficult. It is unclear whether microvascular and myocyte insulin-resistance develop simultaneously. To assess this, we used a rat diet model with a moderate increase (two-fold) in dietary fat. METHODS AND RESULTS Sprague Dawley rats fed normal (4.8% fat wt./wt., 5FD) or high (9.0% fat wt./wt., 9FD) fat diets for 4 weeks were subject to euglycaemic hyperinsulinemic clamp (10 mU/min/kg insulin or saline) or isolated hindlimb perfusion (1.5 or 15 nM insulin or saline). Body weight, epididymal fat mass, and fasting plasma glucose were unaffected by diet. Fasting plasma insulin and non-esterified fatty acid concentrations were significantly elevated in 9FD. Glucose infusion rate and muscle glucose uptake were significantly impaired during insulin clamps in 9FD. Insulin-stimulated microvascular recruitment was significantly blunted in 9FD. Insulin-mediated muscle glucose uptake between 5FD and 9FD were not different during hindlimb perfusion. CONCLUSIONS Impaired insulin-mediated muscle glucose uptake in vivo can be the direct result of reduced microvascular blood flow responses to insulin, and can result from small (two-fold) increases in dietary fat. Thus, microvascular insulin-resistance can occur independently to the development of myocyte insulin-resistance.

Continuous Perfusion Improves Preservation of Donor Rat Hearts: Importance of the Implantation Phase

BACKGROUND Continuous hypothermic perfusion of donor hearts may provide extra protection for long ischemic times and suboptimal donors. The aim of three separate studies was to assess the effect of continuous hypothermic perfusion during simulated donor heart storage and implantation. METHODS In study 1 twelve isolated rat hearts underwent 10 minutes of normothermic ischemia to simulate the effect of brain death on the heart and 5 hours of cardioplegic arrest, using University of Wisconsin solution. Six hearts were statically stored in University of Wisconsin solution at 2 degrees C, and six were perfused with University of Wisconsin solution. To assess the effect of simulated implantation, in study 2 an additional 12 hearts were statically stored for 5.5 hours in University of Wisconsin solution, six of which were rewarmed to a mean of 16 degrees C over the last 30 minutes of arrest. To assess the effect of simulated perfusion, in study 3 during implantation 12 hearts were rewarmed to a mean of 16 degrees C over the last 30 minutes of arrest, during which time six were perfused with 2 degrees C solution. RESULTS Hearts perfused during storage demonstrated greater recovery of prearrest power, 85.8% +/- 1.8%, than hearts preserved by static storage, 72.7% +/- 3.0% (p < 0.01). The simulated warm implantation period reduced recovery of power from 68.3% +/- 5.1% to 40.2% +/- 2.0% (p < 0.001). Perfusion during warm implantation improved recovery to 61.8% +/- 3.9% (p < 0.01). In all experiments improved function was accompanied by improved metabolic energy status. CONCLUSIONS During the implantation period of heart transplantation the donor heart sustains injury that could amount to 50% of total ischemic injury. Continuous perfusion during the cold storage phase and during simulated implantation improves recovery of the donor heart.

Muscle microvascular blood flow responses in insulin resistance and ageing

Insulin resistance plays a key role in the development of type 2 diabetes. Skeletal muscle is the major storage site for glucose following a meal and as such has a key role in maintenance of blood glucose concentrations. Insulin resistance is characterised by impaired insulin‐mediated glucose disposal in skeletal muscle. Multiple mechanisms can contribute to development of muscle insulin resistance and our research has demonstrated an important role for loss of microvascular function within skeletal muscle. We have shown that insulin can enhance blood flow to the microvasculature in muscle thus improving the access of glucose and insulin to the myocytes to augment glucose disposal. Obesity, insulin resistance and ageing are all associated with impaired microvascular responses to insulin in skeletal muscle. Impairments in insulin‐mediated microvascular perfusion in muscle can directly cause insulin resistance, and this event can occur early in the aetiology of this condition. Understanding the mechanisms involved in the loss of microvascular function in muscle has the potential to identify novel treatment strategies to prevent or delay progression of insulin resistance and type 2 diabetes.