Paul Glynn

Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2

Surface fluorescence spectroscopy of the beating heart to measure cytosolic calcium has been limited by the need to use ultraviolet excitation light for many of the commonly used calcium indicators. Ultraviolet light in the heart produces a high level of background fluorescence and is highly absorbed, limiting tissue penetration. Visible wavelength fluorescence dyes such as rhod 2 are available; however, the lack of spectral shift with calcium binding precludes the use of ratio techniques to account for changes in cytosolic dye concentration. We have developed a method for in vivo quantitation of cytosolic rhod 2 concentration that in conjunction with calcium-dependent fluorescence measurements permits estimation of cytosolic calcium levels in perfused rabbit hearts. Reflective absorbance of excitation light by rhod 2 loaded into myocardium was used as an index of dye concentration and the ratio of fluorescence intensity to absorbance as a measure of cytosolic calcium concentration. Endothelial cell loading of rhod 2 was found to be minimal (<5%), and dye leak rate out of the cytosol was slow, with ∼5% loss of dye fluorescence occurring between 10 and 30 min after dye loading. Rhod 2 loading into subcellular compartments, determined by manganese quenching, was also minimal (<5%). The dissociation constant of rhod 2 for calcium was measured in vitro to be 500 nM, and this value increased to 710 nM in the presence of 0.5 mM myoglobin. On the basis of this value and in vivo fluorescence measurements, cytosolic calcium concentration in the rabbit heart was found to be 229 ± 90 nM at end diastole and 930 ± 130 nM at peak systole, with peak fluorescence preceding peak ventricular pressure by ∼40 ms. This technique should facilitate detailed analysis of calcium transients from the whole heart.Surface fluorescence spectroscopy of the beating heart to measure cytosolic calcium has been limited by the need to use ultraviolet excitation light for many of the commonly used calcium indicators. Ultraviolet light in the heart produces a high level of background fluorescence and is highly absorbed, limiting tissue penetration. Visible wave-length fluorescence dyes such as rhod 2 are available; however, the lack of spectral shift with calcium binding precludes the use of ratio techniques to account for changes in cytosolic dye concentration. We have developed a method for in vivo quantitation of cytosolic rhod 2 concentration that in conjunction with calcium-dependent fluorescence measurements permits estimation of cytosolic calcium levels in perfused rabbit hearts. Reflective absorbance of excitation light by rhod 2 loaded into myocardium was used as an index of dye concentration and the ratio of fluorescence intensity to absorbance as a measure of cytosolic calcium concentration. Endothelial cell loading of rhod 2 was found to be minimal (< 5%), and dye leak rate out of the cytosol was slow, with approximately 5% loss of dye fluorescence occurring between 10 and 30 min after dye loading. Rhod 2 loading into subcellular compartments, determined by manganese quenching, was also minimal (< 5%). The dissociation constant of rhod 2 for calcium was measured in vitro to be 500 nM, and this value increased to 710 nM in the presence of 0.5 mM myoglobin. On the basis of this value and in vivo fluorescence measurements, cytosolic calcium concentration in the rabbit heart was found to be 229 +/- 90 nM at end diastole and 930 +/- 130 nM at peak systole, with peak fluorescence preceding peak ventricular pressure by approximately 40 ms. This technique should facilitate detailed analysis of calcium transients from the whole heart.

Vesnarinone and amrinone reduce the systemic inflammatory response syndrome.

OBJECTIVE The systemic inflammatory response is an important cause of organ dysfunction. The present study tested the hypothesis that 2 clinically used agents, amrinone and vesnarinone, would decrease inflammation and cardiac dysfunction in a relevant model of systemic inflammatory response activation. METHODS Rabbits received intravenous endotoxin, alone or in conjunction with amrinone or vesnarinone. Systemic effects were assessed by death, fever, behavior, and acidosis. Measures of inflammatory signaling were (1) plasma tumor necrosis factor-alpha and interleukin-1 beta production, (2) lung tissue myeloperoxidase activity, and (3) myocardial inducible nitric oxide synthase activity. Indices of systolic and diastolic myocardial function were measured in Langendorff-perfused hearts. RESULTS Vesnarinone, in particular, reduced mortality rates (19% vs 61% for lipopolysaccharide alone, P =.01) and acidosis in lipopolysaccharide-treated rabbits. Both agents markedly reduced systemic tumor necrosis factor and interleukin-1 concentrations, lipopolysaccharide-mediated effects on myocardial systolic and diastolic function and on myocardial inducible nitric oxide synthase activity. Vesnarinone, but not amrinone, (1) decreased fever and lethargy, consistent with decreased central nervous system effects of endotoxin, and (2) decreased lung leukocyte infiltration. CONCLUSIONS Vesnarinone and amrinone, which are used clinically for their inotropic and vasodilating properties, may be useful to limit inflammatory activation and consequent organ dysfunction. Structure-activity and/or pharmacokinetic between the compounds may be important, particularly in preventing inflammatory signaling within certain tissues.

ADMINISTRATION OF FRUCTOSE 1,6-DIPHOSPHATE DURING EARLY REPERFUSION SIGNIFICANTLY IMPROVES RECOVERY OF CONTRACTILE FUNCTION IN THE POSTISCHEMIC HEART☆☆☆★

OBJECTIVES Fructose-1,6-diphosphate is a glycolytic intermediate that has been shown experimentally to cross the cell membrane and lead to increased glycolytic flux. Because glycolysis is an important energy source for myocardium during early reperfusion, we sought to determine the effects of fructose-1,6-diphosphate on recovery of postischemic contractile function. METHODS Langendorff-perfused rabbit hearts were infused with fructose-1,6-diphosphate (5 and 10 mmol/L, n = 5 per group) in a nonischemic model. In a second group of hearts subjected to 35 minutes of ischemia at 37 degrees C followed by reperfusion (n = 6 per group), a 5 mmol/L concentration of fructose-1,6-diphosphate was infused during the first 30 minutes of reperfusion. We measured contractile function, glucose uptake, lactate production, and adenosine triphosphate and phosphocreatine levels by phosphorus 31-nuclear magnetic resonance spectroscopy. RESULTS In the nonischemic hearts, fructose-1,6-diphosphate resulted in a dose-dependent increase in glucose uptake, adenosine triphosphate, phosphocreatine, and inorganic phosphate levels. During the infusion of fructose-1,6-diphosphate, developed pressure and extracellular calcium levels decreased. Developed pressure was restored to near control values by normalizing extracellular calcium. In the ischemia/reperfusion model, after 60 minutes of reperfusion the hearts that received fructose-1,6-diphosphate during the first 30 minutes of reperfusion had higher developed pressures (83 +/- 2 vs 70 +/- 4 mm Hg, p < 0.05), lower diastolic pressures (7 +/- 1 vs 12 +/- 2 mm Hg, p < 0.05), and higher phosphocreatine levels than control untreated hearts. Glucose uptake was also greater after ischemia in the hearts treated with fructose-1,6-diphosphate. CONCLUSIONS We conclude that fructose-1,6-diphosphate, when given during early reperfusion, significantly improves recovery of both diastolic and systolic function in association with increased glucose uptake and higher phosphocreatine levels during reperfusion.

Improved Protection of the Hypertrophied Left Ventricle by Histidine-Containing Cardioplegia

BACKGROUND Myocardial hypertrophy has been shown to lead to increased susceptibility to ischemia with accelerated loss of high-energy nucleotides, greater accumulation of H+ and lactate, and earlier onset of contracture. METHODS AND RESULTS To determine whether promoting anaerobic glycolysis during ischemia by buffering H+ results in improved preservation of the hypertrophied heart, we studied the effect of a histidine-containing solution (HBS) on recovery of contractile function and energetic state. Hypertrophied rabbit hearts (aortic banding at 10 days) were subjected to 40 minutes of 37 degrees C ischemia and reperfusion in an isolated Langendorff model. This group was compared with groups receiving St Thomas solution and high-potassium Krebs buffer solution (KCl). Although both phosphocreatine (PCr) and ATP were lower in hypertrophied hearts by end-ischemia compared with nonhypertrophied age-matched controls, there was significantly higher PCr, ATP, and intracellular pH in the HBS group compared with the St Thomas and KCl groups. Recovery of left ventricular developed pressure was best in the HBS group (91% of preischemic values) as was end-diastolic pressure after 30 minutes of reperfusion. Lactate production was also significantly greater in the HBS group, suggesting augmentation of anaerobic glycolysis. CONCLUSIONS We concluded that administration of histidine-containing cardioplegia promotes anaerobic glycolysis and improves recovery of high-energy phosphates and contractile function in hypertrophied myocardium.

Increased myocardial calcium cycling and reduced myofilament calcium sensitivity in early endotoxemia

BACKGROUND Mechanisms of cardiac dysfunction during endotoxemia are multiple and their targets uncertain. This study tested the hypothesis that endotoxin (LPS) induces abnormal calcium-activated contractile force in the heart. METHODS Adult rabbits were given LPS intravenously; 2 hours later hearts were studied in the Langendorff mode. Measurements included peak developed pressure (PDP), myocardial oxygen consumption (MVO2), high-energy phosphates by 31P-NMR, and beat-to-beat intracellular calcium (Cai) by fluorescence spectroscopy. Myofibrillar calcium sensitivity was assessed from the relationship of PDP to Cai and the rate of diastolic Cai removal (tau Ca) was quantified. RESULTS Force-calcium relationships were markedly depressed in LPS hearts despite increased Cai. MVO2 was increased in parallel with increased Cai. Taken together, these data denote myofilament calcium insensitivity and mechanical inefficiency. tau Ca was markedly prolonged in LPS hearts, indicating impaired calcium reuptake and/or extrusion. High-energy phosphates and intracellular pH were unaffected by LPS; however, inorganic phosphate (Pi) was significantly increased. Dobutamine further increased Cai and MVO2 in LPS hearts without significantly improving calcium-activated force. Pyruvate, an inotrope that reduces Pi, significantly improved contractility in LPS hearts. CONCLUSIONS Endotoxemia rapidly induced futile calcium cycling and reduced myofibrillar calcium sensitivity. This state was resistant to beta-agonist inotropic stimulation; inotropes that normalize the calcium-force relationship may be more effective.

Protein Kinase C activation in the heart: Effects on calcium and contractile proteins**

BACKGROUND Cardiac contractile function is dependent on the energetic state of the heart, intracellular calcium levels, and the interaction of the contractile proteins with both adenosine triphosphate and calcium. Protein kinase C (PKC) is a ubiquitous intracellular mediator that has been found in the heart and has been shown to phosphorylate proteins that regulate calcium homeostasis (calcium channels) and the contractile proteins themselves (troponin I and troponin T). METHODS To determine the role of PKC activation on cardiac contractile function, direct activation of PKC was achieved by the infusion of phorbol 12-myristate 13-acetate, an activating phorbol ester. The effects of PKC activation were evaluated in Langendorff-perfused rabbit hearts. Contractile function, high-energy phosphate content (phosphorous-31 nuclear magnetic resonance spectroscopy), oxygen consumption, and intracellular calcium levels (calcium fluorescent dye Rhod-2) were determined. RESULTS Activation of PKC in the heart by phorbol 12-myristate 13-acetate resulted in a significant decrease in both systolic and diastolic function while oxygen consumption and adenosine triphosphate production remained unchanged. Both baseline and peak intracellular calcium levels decreased, which may contribute to the impaired systolic function. CONCLUSIONS Activation of PKC in the heart leads to significant loss of contractile function without affecting energetics. The effect is most likely due to alteration in cytosolic calcium regulation and altered contractile sensitivity to calcium.