Stephen H. Nellis

Small coronary vessel pressure and diameter in an intact beating rabbit heart using fixed-position and free-motion techniques.

We evaluated a technique to measure small vessel pressures and diameters in the right ventricle of a beating rabbit heart. The method allowed free motion of the heart while these measurements were performed. This was accomplished with a strobed light source synchronized with the heart. Thus the observer “sees” the vessels only at the same point in the cardiac cycle. By slowly advancing the Btrobe pulse, we were able to visualize an entire cardiac cycle. We performed pressure measurements with an electromechanical micromanipulator that was programmed to synchronize the motion of a micropipette to the motion of a small vessel. For comparison, another technique was also used which fixed a portion of the right ventricle and allowed motion to be centered around this fixed point. Pressures in veins by our use of the free-motion and fixed techniques were compared and found to be statistically different (6.8 ± 0.7 compared to 13.5 ± 4.7 mm Hg, P< 0.01) with the free-motion technique recording the lower values. The phasic relationship between venous diameter variation and left ventricular pressure also was determined. This relationship was variable between venous networks but quite consistent within a network. The relationship between vessel pressure and vessel diameter revealed significant declines in pressure in relatively large vessels (< 140 μn). This free-motion technique can be used to provide information concerning not only the normal physiology of the coronary circulation, in terms of pressure distributions and the effects of extravascular pressure, but also the changes in vascular pressure which occur in ischemic myocardium and other tissues with inherent motion.

Effects of carnitine in ischemic and fatty acid supplemented swine hearts.

FREE FATTY ACIDS (FFA) IN EXCESS FFA: albumin molar ratios have been determined to additionally compromise mechanical performance in ischemic hearts. Carnitine, an intracellular carrier of FFA and an agent which is lost to the heart during ischemia, has been postulated to in part restore function with its replacement. To test whether its benefits are also operative in a setting of excess FFA, these studies were performed. In the main protocol, four groups of perfused swine hearts (n = 45) were compared during 50 min of control flow (179.7 ml/min) and 40 min of global ischemia (106.1 ml/min). Initial base-line serum FFA:albumin molar ratios and carnitine levels in all groups were 1.3:1 and 8.5 nmol/ml, respectively. In two of these groups FFA:albumin ratios were increased to 5.9:1 with constant infusions of Intralipid. In two alternate groups (one with and one without extra FFA supplements) dl-carnitine was supplied, sufficient to increase serum levels nearly 200-fold. Ischemia per se in 14 hearts significantly decreased several parameters of global and regional mechanical function including left ventricular (LV) and mean aortic pressures, LV isovolumetric pressure development (max dp/dt), LV epicardial motion, and LV work, together with concomitant decreases in myocardial oxygen consumption. Elevated FFA in 12 hearts rendered similarly ischemic further decreased mechanical function (LV pressure: -20.8%, P < 0.05; mean aortic pressure -26.9%, P < 0.05; LV max dp/dt: -39%, P < 0.05; regional LV shortening: -51.1%, P < 0.05; and LV work: -50.3%, P < 0.05) as compared with nonsupplemented hearts. dl-Carnitine treatments in nine hearts, not supplemented with extra FFA were without apparent effect in improving overall hemodynamic performance. However, dl-carnitine in 10 high FFA-ischemic hearts effected several improvements as compared with the untreated group: LV pressure was increased 25.6%, P < 0.025; mean aortic pressure: +43.5%, P < 0.05; LV max dp/dt: +41.5%, P < 0.05; regional LV shortening: +241.3%, P < 0.001; and LV work: +76.2%, P < 0.05 at comparable levels of myocardial oxygen consumption. In a separate protocol, the effects of stereospecificity were also studied by comparing l- with dl-carnitine in globally perfused, palmitate-supplemented hearts (five hearts in each treatment group). At similar conditions of flow and serum FFA, changes in mechanical function were comparable, except for a tendency to perform greater LV work at reduced flows in the l-carnitine-treated hearts. Thus, it was demonstrated that carnitine in ischemic hearts is capable of preserving mechanical function under conditions of excess FFA, presumably by modifying the toxic effects of FFA intermediates. The major therapeutic actions appeared to derive from the l-isomer of carnitine.

Mechanical and metabolic functions in pig hearts after 4 days of chronic coronary stenosis

OBJECTIVES This study sought to evaluate the functional and metabolic consequences of imposing a chronic external coronary stenosis around the left anterior descending coronary artery for 4 days in an intact pig model. BACKGROUND A clinical condition termed hibernating myocardium has been described wherein as a result of chronic sustained or intermittent coronary hypoperfusion, heart muscle minimizes energy demands by decreasing mechanical function and thus avoids cell death. The use of chronic animal models to stimulate this disorder may assist in establishing causative associations among determinants to explain this phenomenon. METHODS A hydraulic cuff occluder was placed around the left anterior descending coronary artery in eight pigs. Coronary flow velocity was reduced by a mean (+/- SE) of 49 +/- 5% of prestenotic values, as estimated by a Doppler velocity probe. After 4 days the pigs were prepared with extracorporeal coronary circulation and evaluated at flow conditions dictated by the cuff occluder. Substrate utilizations were described using equilibrium labeling with [U-14C]palmitate and [5-3H]glucose. Results were compared with a combined group of 21 acute and chronic (4 day) sham animals. RESULTS Four days of partial coronary stenosis significantly decreased regional systolic shortening by 54%. Myocardial oxygen consumption was maintained at aerobic levels, and rest coronary flows were normal. Fatty acid oxidation was decreased by 43% below composite sham values, and exogenous glucose utilization was increased severalfold. Alterations in myocardial metabolism were accompanied by a decline in tissue content of adenosine triphosphate. CONCLUSIONS These data suggest that chronic coronary stenosis in the absence of macroscarring imparts an impairment in mechanical function, whereas coronary flow and myocardial oxygen consumption are preserved at rest. The increases in glycolytic flux of exogenous glucose are similar to observations on glucose uptake assessed by fluorine-18 2-deoxy-2-fluoro-D-glucose in patients with advanced coronary artery disease. We speculate that intermittent episodes of ischemia and reperfusion are the cause of this phenomenon.

Effects of carnitine isomers on fatty acid metabolism in ischemic swine hearts.

We studied the hemodynamic and metabolic effects of treatments with the L- and DL- isomers of carnitine in four groups (n = 42) of intact working swine hearts rendered mildly ischemic (−46% reduction in global perfusion). In three groups (n = 34), free fatty acids (FFA) in the coronary perfusate were augmented with labeled palmitate (0.72 /μnol/ml). The inclusion of excess FFA per se (n = 11), as compared with nonsupplemented hearts, further and significantly depressed mechanical function (−40% and −55% declines in left ventricular (LV)-developed pressure and work), stimulated a 2-fold increase in FFA uptake (but without a corresponding increase in “CO2 production), raised fatty acyl-CoA levels in tissue (94 to 132 nmol/g dry), and shortened group survival. Treatments with both L- and DL-carnitine (serum values 6687 and 6398 nmol/ml) effected significant improvements in several parameters of mechanical function and survival. LV-developed pressure and work at end-ischemia were increased (+50% and +62% in L-treated hearts and +48% and +65% in DL-treated hearts). Myocardial oxygen consumption was similar to that of untreated FFA-supplemented hearts. During the middle to late portion of the perfusion trials in L-treated hearts (n = 9), FFA uptake and14CO2production were significantly reduced, and accumulation of tissue long-chain acyl-CoA was less (−44%, P < 0.05). Metabolic trends in DL-carnitine-treated hearts ranged between those in untreated and L- carnitine-treated hearts. These data suggest that carnitine reduces availability or incorporation of FFA intracellularly, and this benefits the heart mechanically during ischemic restrictions in coronary flow. The L-isomer appears to be the more biologically active.

Effects of blunt cardiac trauma on coronary vasomotion, perfusion, myocardial mechanics, and metabolism.

: Blunt injury to heart muscle can result in a variety of dysrhythmias and mechanical dysfunction. In the present studies of 24 open-chest, working swine hearts with controlled perfusion of the left anterior descending (LAD) coronary artery, changes in proximal and distal coronary vascular resistance (CVR), small-vessel perfusion (using radioactively-labeled microspheres), and regional and global mechanical function and metabolism (myocardial oxygen consumption [MVO2] and lactate extraction) were observed before and for 1 hour following a single impact involving the LAD artery. Trauma caused no spasm, thrombosis, hemorrhage, or laceration of the LAD artery but resulted in significant perfusion redistributions of small vessels. Within minutes of the impact, epicardial/endocadial flow ratios in the myocardial tissue perfused by the traumatized vessel increased (p < 0.005) and were associated with a significant decrease in the distal CVR (p < 0.001). In this same region, significant decreases were also observed in an index of regional work (p < 0.01), shortening (p < 0.005), MVO2 (p < 0.001), and per cent lactate extraction (p < 0.01). Also noted were declines in left ventricular (LV) pressure development and contractility (LV max dp/dt). The regional changes in flow patterns and function in general persisted throughout the course of perfusion. These data suggest that cardiac trauma can induce major changes in vasomotor tone and perfusion distributions of the coronary vasculature, and demonstrate how blunt cardiac-coronary trauma can result in some of the hemodynamic and electrocardiographic abnormalities previously reported.Blunt injury to heart muscle can result in a variety of dysrhythmias and mechanical dysfunction. In the present studies of 24 open-chest, working swine hearts with controlled perfusion of the left anterior descending (LAD) coronary artery, changes in proximal and distal coronary vascular resistance (CVR), small-vessel perfusion (using radioactively-labeled microspheres), and regional and global mechanical function and metabolism (myocardial oxygen consumption [MVO2] and lactate extraction) were observed before and for 1 hour following a single impact involving the LAD artery. Trauma caused no spasm, thrombosis, hemorrhage, or laceration of the LAD artery but resulted in significant perfusion redistributions of small vessels. Within minutes of the impact, epicardial/endocadial flow ratios in the myocardial tissue perfused by the traumatized vessel increased (p < 0.005) and were associated with a significant decrease in the distal CVR (p < 0.001). In this same region, significant decreases were also observed in an index of regional work (p < 0.01), shortening (p < 0.005), MVO2 (p < 0.001), and per cent lactate extraction (p < 0.01). Also noted were declines in left ventricular (LV) pressure development and contractility (LV max dp/dt). The regional changes in flow patterns and function in general persisted throughout the course of perfusion. These data suggest that cardiac trauma can induce major changes in vasomotor tone and perfusion distributions of the coronary vasculature, and demonstrate how blunt cardiac-coronary trauma can result in some of the hemodynamic and electrocardiographic abnormalities previously reported.

Correlation between [5-3H]glucose and [U-14C]deoxyglucose as markers of glycolysis in reperfused myocardium.

Studies were conducted in extracorporeally perfused, intact, working pig hearts to determine whether, in heart muscle, trace-labeled deoxyglucose serves as an accurate marker of glycolytic flux in reperfusion after exposures to mild to moderate regional ischemia. In the main study, two groups of hearts were compared, as distinguished by levels of glucose in the whole-blood perfusate (euglycemic hearts [group I], blood glucose of 7.4 +/- 0.2 mumol/ml, n = 7; hyperglycemic hearts [group II], blood glucose of 12.9 +/- 0.5 mumol/ml, n = 8). Both groups were subjected to a 60% reduction in anterior descending coronary flow for 30 minutes followed by reperfusion for 40 minutes. Modest and comparable regional mechanical stunning during reflow was noted in both groups. Glucose utilization, as estimated from the release of 3H2O from the steady-state infusion of [5-3H]glucose during aerobic perfusion, was modest but during reperfusion was noted to increase significantly above aerobic values in each of the two groups, with a doubling of rates in group II hearts compared with group I hearts (p less than 0.041 or p less than 0.090). Net lactate extraction was comparable in reflow in both groups, suggesting in this specific instance a preferential enhancement of glucose oxidation in hyperglycemic group II hearts. Shifts in accumulation of tissue radioactivity of [U-14C]2-deoxyglucose in reperfused myocardium were not able to track these trends. The variability of 14C-labeled radioactivity among animals was marked and essentially masked any ability to discern trends in glycolysis as described by tritiated glucose between the aerobic and reperfusion intervals. When the data were arrayed by linear regression analysis, the slopes derived from 14C-labeled deoxyglucose were either discordant or insensitive to those described by 3H-labeled glucose. Tissue glycogen levels were slow to recover in early reflow and at end reperfusion were still significantly depressed from aerobic levels. The present data indicate that coronary reperfusion and hyperglycemia have influence in determining glycolytic flux in myocardium. Labeled deoxyglucose, considered solely as a marker of exogenous glucose utilization, appears to be an insensitive agent in describing these events at conditions of relatively low glucose flux.

Distribution of carbon flux within fatty acid utilization during myocardial ischemia and reperfusion.

Twenty-nine intact, working pig hearts were extracorporeally perfused and divided into two study groups (16 Aerobic and 13 Ischemic/Reflow hearts). Step function, equilibrium labeling with [14C]palmitate was used to develop uptake and washout curves of radioactive fatty acid products contained in coronary effluent during either aerobic perfusion or reperfusion after ischemia (60% reduction in left anterior descending coronary flow for 30 minutes). Left anterior descending control flows were slightly overperfused in Aerobic hearts (18% higher than in Ischemic/Reflow hearts); otherwise, circumflex and right coronary flows, left ventricular pressure, and serum fatty acids and blood sugar levels were comparable between groups. As expected in Ischemic/Reflow hearts, recovery of regional systolic shortening and myocardial oxygen consumption in reperfusion was only modestly impaired (-20% and -19%, respectively, not significant and p less than 0.011 compared with preischemic values, not significant from Aerobic hearts). The only significant metabolized product to be released from labeled fatty acid utilization in either group was 14CO2. A smaller fatty acid pool also was measured and accounted for by that contained in the coronary intravascular volume. We could determine no significant back diffusion of fatty acids from myocardium in either perfusion condition. Uptake time constants of the early phase of 14CO2 production also were virtually identical in both groups (19.9 +/- 3.2 versus 16.7 +/- 3.2 minutes in Aerobic and Ischemic/Reflow hearts, respectively) and strongly correlated with hemodynamics as described by heart rate. In washout studies, tissue radioactivity in the aqueous soluble and fatty acid pools declined in both study groups, and counts in complex lipids and cholesterol/cholesteryl esters remained steady, whereas those in triacylglycerols varied. Washout of 14CO2 in both groups never reached background radioactivity over a 40-minute sampling after cessation of isotope infusion into the perfusate, suggesting slow release of trapped substrate from intracellular pools, which then proceeded to fatty acid oxidation. In conclusion, these experiments have demonstrated very similar findings with respect to fatty acid uptake, storage, and release characteristics between aerobic and reperfused myocardium. We found no differences in preferred substrate utilization and oxidation as a result of reversible ischemia followed by reflow.

The role of glucose metabolism in a pig heart model of short-term hibernation

Previously, we reported, alterations in glucose metabolism in a 4 day model of chronic coronary stenosis similar to those described in patients with hibernating hearts. The purpose of this study was 2 fold: (1) to identify whether an acute model of mild, sustained ischemia could effect similar changes, and (2) to determine the effects of pharmacological inhibition of glycolysis. In the first group, extracorporeally perfused, intact pig hearts were subjected to 85 min of a 40% reduction in left anterior descending (LAD) coronary arterial blood flow. A second group was subjected to the same protocol, except after 40 min of LAD regional ischemia, iodoacetate (IAA) was administered to block glycolysis. Ischemia reduced MVO2 by 10% in both groups with a further 20% reduction noted following IAA treatment. Regional systolic shortening was reduced nearly 50% by ischemia and decreased an additional 40% following treatment with IAA. Glycolysis was increased by over 700% with ischemia in the first group. IAA caused a 3 fold reduction in glycolysis as compared to the preceding ischemic period and inhibited lactate production. Fatty acid metabolism was significantly reduced by ischemia in the first group, but was not reduced in the IAA group. Activity of creatine kinase associated with myofibrils was reduced and may have contributed to the contractile dysfunction. In conclusion, this acute model of short-term hibernation demonstrates several metabolic changes previously reported in chronic hibernation and may prove useful in determining mechanisms of substrate utilization in simulated conditions of chronic coronary stenosis and hibernation.

The effects of norepinephrine on active hyperemia in the canine gracilis muscle.

We studied the effects of intra-arterial norepinephrine (NE) on skeletal muscle blood flow (BF), oxygen consumption (VOi), and arteriovenous oxygen difference (A-Voj) at rest and during exercise in an autoperfused canine gracilis muscle preparation.Static continuous exercise at a fixed level of maximal developed tension (P6) was induced by gracilis nerve stimulation; developed tension was monitored and used to control stimulation voltage. In one group of dogs (n − 10), data were collected before (rest) and at the end of each of a series of four 2-minute periods of exercise (10% P) in each preparation. During both the rest and the exercise phases, continuous intra-arterial infusions of isotonic saline alone (control) and saline plus NE (0.11, 0.22, and 0.44 pg/min) were made. Control resting data were: BF-5.90 ml/min; A-VOi-5.30 vol %; Voi-0.31 ml/min. NE during rest reduced BF by 39-69%, increased A-VOi by 79-91%, and reduced VOi by an average of 41.9%. Control exercise data were: BF-17.2 ml/min; A-VO2 = 11.2 vol %; Voi-1.96 ml/min. NE during exercise attenuated BF by 7-65% and widened A-VOi by 22-35%. Vo2 was maintained at control exercise levels during lower NE infusion levels but was attenuated by 56% at the highest NE level. In the second group of dogs (n-8), data were collected at rest and at four times during a 10-minute exercise period (2.5% Po). NE (0.089, 0.17, and 0.34 pg/min) or saline (control saline) was infused for 2 minutes each during the final 7 minutes of exercise. At the lower NE doses, no significant difference was observed relative to the control-saline experiment At the highest NE dose BF and VOi were attenuated (BF: −22%, Vo,: −20%), and A-VOi was unchanged compared to control. The NE-induced attenuation in BF and Voj during exercise may in part result from a mechanism similar to that which occurs in congestive heart failure in which an exaggerated sympathoadrenal response during exercise and an attenuated exerciseinduced rise in forearm Vd occurs. Ore Ret 44: 680-666, 1979

Influence of Myocardial Contraction on Coronary Microcirculation: Techniques and Results

For several years, our laboratory has been examining the hemodynamics of the coronary microcirculation with particular emphasis on the effect of myocardial contraction on these small vessels. The initial goals of the laboratory were to measure flow, diameter, and pressure in small vessels and relate the phasic characteristics of these measurements to each other.