The purpose of this study was to develop Ull isoluted, pulsatile blood-perfused fut lung model th... more The purpose of this study was to develop Ull isoluted, pulsatile blood-perfused fut lung model that allows us to evuluatc the preserved lung functions. Lungs isolated from Sprague-Duwlcy rats were perfused with venous whole blood by either a pulsatile or constant flm\-', The effluent wus COIltinuously deoxygenated with u 95% Ni5% CO 2 gas mixture. Ainmy resistance, hmg compliance, clastic work, flow resistive work, pulmonary vascular resistance, and blood gas analysis were assessed. Pressor responses to NG-monomethyl-L-arginine (L-NMMA) were compared between pulsatile and constant blood flow. At n flow of 0.1 mUg body weight/min, pulsatile perfusion allowed for stable perfusion at least for 2 It (mean 162.5 ± 15.1min) with stable aerodynamic and hemodynamic variables including blood gas tellsions, whereas COIIstnnt perfusion resuUed in immediute lung failure. When L-NMMA was added to the perfusate, the mean pulmonary artery pressure did not show any change in the constant flow (6.0 :!:: 2.6% increase), but did show a significant increase in the pulsatile lIow (45 ± 11% increase). Pulsatile blood flow reduced the pulmonary vascular resistance relative to the constnnt flow and allowed for a 2-11 perfusion period to evaluate the lung function. The vasorelaxant mechanism in the pulsatile perfusion is reluted in part to the endothelial-dependent relaxation observed in the nitric oxide pathwny.
Several cellular and molecular mechanisms have been implicated in the development of myocardial d... more Several cellular and molecular mechanisms have been implicated in the development of myocardial dysfunction and low cardiac output in pediatric patients undergoing heart surgery. Ischemia-reperfusion injury with alterations in calcium homeostasis as well as mitochondrial function has been strongly related to myocyte damage and heart failure in this population. In this article, we will review the main mechanisms of postoperative cardiac dysfunction at a cellular and molecular level and the subsequent protective strategies. In addition, we will describe cellular features of the neonatal or immature myocardium and will suggest possible protective management strategies. This article addresses the first of eight topics comprising the special issue entitled "Pharmacologic strategies with afterload reduction in low cardiac output syndrome after pediatric cardiac surgery".
... Our studies, and those of others, have demonstrated that alterations in mitochondrial structu... more ... Our studies, and those of others, have demonstrated that alterations in mitochondrial structure and function occur during ischemia and extend into reperfusion to severely compromise post-ischemic functional recovery and cellular viability (Faulk et al. 1995a; Lesnefsky et al. ...
Magnesium research : official organ of the International Society for the Development of Research on Magnesium, 1997
Studies examining the effects of aging on the myocardium have indicated that with advancing age t... more Studies examining the effects of aging on the myocardium have indicated that with advancing age there are anatomical, mechanical, ultrastructural, and biochemical alterations which compromise the adaptive response of the heart and make the senescent myocardium less tolerant to surgically-induced ischemia. With the increased incidence of elderly patients as candidates for complex cardiac surgery, the investigation into methods which will increase survivability and enhance myocardial protection are of paramount importance. Previous reports have indicated that surgically-induced global ischemia is associated with alteration in cytosolic calcium accumulation ([Ca2+]i), and that the level of post-ischemic functional recovery can be correlated with control of [Ca2+]i Cardioplegia (high potassium arrest) partially ameliorates the adverse effects associated with global ischemia however; the use of magnesium (magnesium supplemented potassium icardioplegia) has been shown to provide superior ...
Human ascending thoracic aortic aneurysms (ATAAs) are life threatening and constitute a leading c... more Human ascending thoracic aortic aneurysms (ATAAs) are life threatening and constitute a leading cause of mortality in the United States. Previously, we demonstrated that collagens α2(V) and α1(XI) mRNA and protein expression levels are significantly increased in ATAAs. In this report, the authors extended these preliminary studies using high-throughput proteomic analysis to identify additional biomarkers for use in whole blood real-time RT-PCR analysis to allow for the identification of ATAAs before dissection or rupture. Human ATAA samples were obtained from male and female patients aged 65 ± 14 years. Both bicuspid and tricuspid aortic valve patients were included and compared with nonaneurysmal aortas (mean diameter 2.3 cm). Five biomarkers were identified as being suitable for detection and identification of ATAAs using qRT-PCR analysis of whole blood. Analysis of 41 samples (19 small, 13 medium-sized, and 9 large ATAAs) demonstrated the overexpression of 3 of these transcript b...
American journal of physiology. Heart and circulatory physiology, 2000
Adenosine-enhanced ischemic preconditioning (APC) extends the protection afforded by ischemic pre... more Adenosine-enhanced ischemic preconditioning (APC) extends the protection afforded by ischemic preconditioning (IPC) by both significantly decreasing infarct size and significantly enhancing postischemic functional recovery. The purpose of this study was to determine whether APC is modulated by ATP-sensitive potassium (K(ATP)) channels and to determine whether this modulation occurs before ischemia or during reperfusion. The role of K(ATP) channels before ischemia (I), during reperfusion (R), or during ischemia and reperfusion (IR) was investigated using the nonspecific K(ATP) blocker glibenclamide (Glb), the mitochondrial (mito) K(ATP) channel blocker 5-hydroxydecanoate (5-HD), and the sarcolemmal (sarc) K(ATP) channel blocker HMR-1883 (HMR). Infarct size was significantly increased (P < 0.05) in APC hearts with Glb-I, Glb-R, and 5-HD-I treatment and partially with 5-HD-R. Glb-I and Glb-R treatment significantly decreased APC functional recovery (P < 0.05 vs. APC), whereas 5-H...
Previously, we have shown that potassium and magnesium (K-Mg, 20 mM each) cardioplegia ameliorate... more Previously, we have shown that potassium and magnesium (K-Mg, 20 mM each) cardioplegia ameliorated cytosolic calcium ([Ca2+]i) accumulation and was associated with enhanced functional recovery after surgically induced global ischemia in the aged heart. K-Mg cardioplegia was also shown to enhance cytosolic cytochrome oxidase I activity and mRNA levels, suggesting that enhanced functional recovery may involve the preservation of high-energy phosphates. To investigate this hypothesis, 31P nuclear magnetic resonance was used to measure serial alterations in phosphocreatine (PCr), inorganic phosphate, nucleoside triphosphate (NTP), intracellular free magnesium (Mgf), and intracellular pH (pHi) in Langendorff-perfused, aged (135 wk) rabbit hearts during preischemia, global ischemia (30 min), and reperfusion (30 min). K-Mg cardioplegia retarded PCr depletion (P < 0.05) and significantly enhanced NTP preservation (P < 0.05) during ischemia and reperfusion. K-Mg cardioplegia also atten...
Cytosolic calcium in the myocardium is rapidly accumulated during ischemia and has been correlate... more Cytosolic calcium in the myocardium is rapidly accumulated during ischemia and has been correlated with the attenuation of functional recovery in the myocardium. The aged myocardium is more sensitive to ischemia and accumulates significantly more cytosolic calcium than either the newborn or the mature myocardium. Modification of the age-related propensity to increased cytosolic calcium accumulation may be achieved through the use of magnesium or potassium/magnesium cardioplegia. Improved postischemic ventricular function obtained with magnesium or potassium/magnesium cardioplegia may have important implications in the reduction of myocardial morbidity and mortality.
Both heat-shock protein (HSP) 70 and its mRNA are induced in the ischemic myocardium. The inducto... more Both heat-shock protein (HSP) 70 and its mRNA are induced in the ischemic myocardium. The inductor stimuli are, however, not known. Stretch and ischemic metabolic alterations are the two most likely HSP70 inductors. To assess whether anaerobic metabolism induces HSP70 mRNA expression, 61 rat hearts were perfused with Krebs-Henseleit buffer (37 degrees C) in a modified Langendorff apparatus built for rapid switching between pressure-controlled and flow-controlled perfusions. Each heart was initially perfused for 30 minutes at a constant perfusion pressure of 65 mm Hg. Subsequently, separate hearts were perfused at a constant flow of 8, 6, 4, 2, 1, 0.5, or 0 mL/min for 30 minutes using a nonpulsatile pump (n = 5, each perfusion level). Hemodynamic measurements demonstrated a linear correlation between coronary flow and both perfusion pressure and developed pressure (r = .94 and r = .89, respectively; P < .0001 for both comparisons). Diastolic pressure at the end of the perfusions i...
We carried out a study to relate the effect of the type of dietary fat and ethanol on antioxidant... more We carried out a study to relate the effect of the type of dietary fat and ethanol on antioxidant enzyme mRNA levels in liver in the intragastric feeding rat model. Different types of dietary fat were administered [saturated fat (SE), corn oil (CE) and fish oil (FE)] with ethanol to induce varying severities of liver injury. Ethanol-fed rats were pair-fed with dextrose-fed controls that received isocaloric amounts of dextrose. All animals were killed at 1 month and the following studies were carried out: evaluation of severity of pathologic liver injury, mRNA quantitation for catalase, glutathione peroxidase (GPx), and manganese superoxide dismutase (MnSOD), microsomal conjugated dienes, and hydrogen peroxide. SE animals had no liver injury, FE animals had severe liver injury, and CE animals had moderate liver injury. Ethanol induced GPx mRNA in all dietary groups, with the highest levels seen in the FE group. The pattern of catalase mRNA induction was similar to that of GPx mRNA. I...
Background: We examined the effects of in vivo administration of a potent inhibitor of iron-depen... more Background: We examined the effects of in vivo administration of a potent inhibitor of iron-dependent lipid peroxidation, lazaroid U74389G, on lung preservation.
Previously described mitochondrial isolation methods using differential centrifugation and/or Fic... more Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 10(10) viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.
ABSTRACT The purpose of this study was to develop an isolated, pulsatile blood-perfused rat lung ... more ABSTRACT The purpose of this study was to develop an isolated, pulsatile blood-perfused rat lung model that allows us to evaluate the preserved lung functions. Lungs isolated from Sprague-Dawley rats, were perfused with venous whole blood by either a pulsatile or constant flow. The effuent was continuously deoxygenated with a 95% N2/5% CO2 gas mixture. Airway resistance, lung compliance, elastic work, flow resistive work, pulmonary vascular resistance, and blood gas analysis were assessed. Pressor responses toN G -monomethyl-l-arginine (l-NMMA) were compared between pulsatile and constant blood flow. At a flow of 0.1 ml/g body weight/min, pulsatile perfusion allowed for stable perfusion at least for 2h (mean 162.5±15.1 min) with stable aerodynamic and hemodynamic variables including blood gas tensions, whereas constant perfusion resulted in immediate lung failure. Whenl-NMMA was added to the perfusate, the mean pulmonary artery pressure did not show any change in the constant flow (6.0±2.6% increase), but did show a significant increase in the pulsatile flow (45±11% increase). Pulsatile blood flow reduced the pulmonary vascular resistance relative to the constant flow and allowed for a 2-h perfusion period to evaluate the lung function. The vasorelaxant mechanism in the pulsatile perfusion is related in part to the endothelial-dependent relaxation observed in the nitric oxide pathway.
1 other HighWire hosted article: This article has been cited by [PDF] [Full Text] [Abstract] , No... more 1 other HighWire hosted article: This article has been cited by [PDF] [Full Text] [Abstract] , November 1, 2010; 88 (2): 211-218. Cardiovasc Res M. Gucek and E. Murphy What can we learn about cardioprotection from the cardiac mitochondrial proteome?
The Journal of Thoracic and Cardiovascular Surgery, 1996
Objective: The effect of cardioplegic solutions with high concentrations of potassium or magnesiu... more Objective: The effect of cardioplegic solutions with high concentrations of potassium or magnesium (or both) on cytosolic calcium accumulation was investigated with fura-2 in isolated perfused mature (n = 24) and aged (n = 24) rabbit hearts. Methods: We compared cytosolic calcium accumulation before ischemia (control), during 30 minutes of ischemia and 30 minutes of reperfusion under global ischemia, or after treatment with potassium (20 mmol/L), magnesium (20 retool/L), or both. Results: Cytosolic calcium accumulation was increased during global ischemia in the mature heart (from 178.7 +-. 24.2 in the control group to 393.6 +-25.5 nmol/L; p < 0.005) and in the aged heart (from 187.4 + 18.7 in the control group to 501.0 +-46.1 nmol/L; p < 0.005). Potassium reduced cytosolic calcium accumulation during ischemia in both the mature and aged hearts (300.9 _ 23.2 and 365.2 -27.7 nmol/L, respectively; p < 0.05 vs global ischemia). Magnesium and potassium/magnesium completely controlled cytosolic calcium accumulation in the mature heart (198.7 +-27.5 nmol/L; p < 0.01 vs global ischemia andp < 0.05 vs potassium: 182.3 _+ 22.7 nmol/L;p < 0.05 vs global ischemia and potassium, respectively). Magnesium and potassium/magnesium attenuated cytosolic calcium accumulation in the aged heart (261.3 -+ 26.7, 262.3 +-. 25.2 nmol/L, respectively; p < 0.01 vs global ischemia). These changes in cytosolic calcium accumulation correlated with improved postischemic ventricular function. To investigate the mechanism(s) of magnesium-supplemented cardioplegic inhibition of cytosolic calcium accumulation, we performed parallel studies (n = 43) using nifedipine, ryanodine, and dimethylthiourea. Nifedipine with or without ryanodine reduced eytosolic calcium accumulation. Dimethylthiourea did not alter cytosolic calcium accumulation during global ischemia. Our results suggest that eytosolic calcium accumulation during global ischemia was mainly increased via the sarcolemmal 1-type calcium channel and the sarcoplasmic reticulum calcium-release channel. The modulating action of potassium/ magnesium cardioplegia on cytosolic calcium accumulation during ischemia would appear to act through the inhibition of the myocardial 1-type calcium channel and the sarcoplasmic reticulum calcium-release channel. Conclusion: Senescent cardiac dysfunction correlates with increased ischemia-induced cytosolic calcium accumulation. Magnesium-supplemented potassium cardioplegia ameliorates this age-related phenomenon at normothermia and may have important implications in myocardial protection in the elderly population. (J Thorac Cardiovasc Surg 1996;112:175-84) From the Division
We have established a highly sensitive functional screen for the isolation of cDNAs encoding cAMP... more We have established a highly sensitive functional screen for the isolation of cDNAs encoding cAMP phosphodiesterases (PDEs) by complementation of defects in a Saccharomyces cereuisiae strain lacking both endogenous cAMP PDE genes, PDEl and PDE2. Three groups of cDNAs corresponding to three distinct human genes encoding CAMP-specific PDEs were isolated from a human glioblastoma cDNA library using this functional screen. Two of these genes are closely related to the Drosophila dunce CAMP-specific PDE. The third gene, which we named HCP1, encoded a novel CAMP-specific PDE. HCPl has an amino acid sequence related to the sequences of the catalytic domains of all cyclic nucleotide PDEs. HCPl is a high affinity CAMPspecific PDE (K, = 0.2 pM) that does not share other properties of the CAMP-specific PDE family, i.e. extensive sequence homology to the Drosophila dunce cAMP PDE and sensitivity to rolipram and R020-1724. The PDE activity of HCPl is not sensitive to cGMP or other inhibitors of the cGMP-inhibitable PDEs, such as milrinone. The biochemical and pharmacological properties of HCPl suggest that it is a member of a previously undiscovered cyclic nucleotide PDE family. Northern blot analysis indicates that high levels of HCP 1 mRNA are present in human skeletal muscle.
Journal of Molecular and Cellular Cardiology, 1987
A system for RNA transcription in vitro was established in order to determine the relative rate o... more A system for RNA transcription in vitro was established in order to determine the relative rate of RNA synthesis in neonatal and adult rat myocardial cells. This assay system optimizes the incorporation of [3H]UMP into RNA by using 3.5 x 107 myocardial-cell nuclei, and minimizes RNA degradation for at least 1 h in transcription in vitro, by the addition of human placental RNAase inhibitor. A 1000 increase in the incorporation of [3H]UMP into myocardial-cell RNA was found on addition of this inhibitor. Myocardialcell nuclei derived from 5-, 10-, 15-, 20-, and > 100-day-old rat hearts indicated that there is a progressive decrease in RNA synthesis with age. A 3-fold increase in RNA synthesis in 5-day-old myocardial cell nuclei as compared with 20-day-old rat heart was found. RNA synthesis in the adult myocardial cell nuclei decreased more than 10-fold in comparison with the 5-day-old newborn. The incorporation of [3H]UMP into rat liver nuclear RNA was 3-fold greater than in the myocardial-cell nuclear RNA, even when compared with the highly active transcription of 12-day-old heart nuclei. In order to determine the relationship between total RNA synthesis and the extent of specific gene expression in myocardial-cell nuclei during development, two distinct cDNA probes were used for Northern-blot analysis. Our results indicate that myosin-heavy-chain gene expression is remarkably decreased with age, whereas the 'housekeeping' gene is continually expressed independently of age.
The purpose of this study was to develop Ull isoluted, pulsatile blood-perfused fut lung model th... more The purpose of this study was to develop Ull isoluted, pulsatile blood-perfused fut lung model that allows us to evuluatc the preserved lung functions. Lungs isolated from Sprague-Duwlcy rats were perfused with venous whole blood by either a pulsatile or constant flm\-', The effluent wus COIltinuously deoxygenated with u 95% Ni5% CO 2 gas mixture. Ainmy resistance, hmg compliance, clastic work, flow resistive work, pulmonary vascular resistance, and blood gas analysis were assessed. Pressor responses to NG-monomethyl-L-arginine (L-NMMA) were compared between pulsatile and constant blood flow. At n flow of 0.1 mUg body weight/min, pulsatile perfusion allowed for stable perfusion at least for 2 It (mean 162.5 ± 15.1min) with stable aerodynamic and hemodynamic variables including blood gas tellsions, whereas COIIstnnt perfusion resuUed in immediute lung failure. When L-NMMA was added to the perfusate, the mean pulmonary artery pressure did not show any change in the constant flow (6.0 :!:: 2.6% increase), but did show a significant increase in the pulsatile lIow (45 ± 11% increase). Pulsatile blood flow reduced the pulmonary vascular resistance relative to the constnnt flow and allowed for a 2-11 perfusion period to evaluate the lung function. The vasorelaxant mechanism in the pulsatile perfusion is reluted in part to the endothelial-dependent relaxation observed in the nitric oxide pathwny.
Several cellular and molecular mechanisms have been implicated in the development of myocardial d... more Several cellular and molecular mechanisms have been implicated in the development of myocardial dysfunction and low cardiac output in pediatric patients undergoing heart surgery. Ischemia-reperfusion injury with alterations in calcium homeostasis as well as mitochondrial function has been strongly related to myocyte damage and heart failure in this population. In this article, we will review the main mechanisms of postoperative cardiac dysfunction at a cellular and molecular level and the subsequent protective strategies. In addition, we will describe cellular features of the neonatal or immature myocardium and will suggest possible protective management strategies. This article addresses the first of eight topics comprising the special issue entitled &amp;amp;quot;Pharmacologic strategies with afterload reduction in low cardiac output syndrome after pediatric cardiac surgery&amp;amp;quot;.
... Our studies, and those of others, have demonstrated that alterations in mitochondrial structu... more ... Our studies, and those of others, have demonstrated that alterations in mitochondrial structure and function occur during ischemia and extend into reperfusion to severely compromise post-ischemic functional recovery and cellular viability (Faulk et al. 1995a; Lesnefsky et al. ...
Magnesium research : official organ of the International Society for the Development of Research on Magnesium, 1997
Studies examining the effects of aging on the myocardium have indicated that with advancing age t... more Studies examining the effects of aging on the myocardium have indicated that with advancing age there are anatomical, mechanical, ultrastructural, and biochemical alterations which compromise the adaptive response of the heart and make the senescent myocardium less tolerant to surgically-induced ischemia. With the increased incidence of elderly patients as candidates for complex cardiac surgery, the investigation into methods which will increase survivability and enhance myocardial protection are of paramount importance. Previous reports have indicated that surgically-induced global ischemia is associated with alteration in cytosolic calcium accumulation ([Ca2+]i), and that the level of post-ischemic functional recovery can be correlated with control of [Ca2+]i Cardioplegia (high potassium arrest) partially ameliorates the adverse effects associated with global ischemia however; the use of magnesium (magnesium supplemented potassium icardioplegia) has been shown to provide superior ...
Human ascending thoracic aortic aneurysms (ATAAs) are life threatening and constitute a leading c... more Human ascending thoracic aortic aneurysms (ATAAs) are life threatening and constitute a leading cause of mortality in the United States. Previously, we demonstrated that collagens α2(V) and α1(XI) mRNA and protein expression levels are significantly increased in ATAAs. In this report, the authors extended these preliminary studies using high-throughput proteomic analysis to identify additional biomarkers for use in whole blood real-time RT-PCR analysis to allow for the identification of ATAAs before dissection or rupture. Human ATAA samples were obtained from male and female patients aged 65 ± 14 years. Both bicuspid and tricuspid aortic valve patients were included and compared with nonaneurysmal aortas (mean diameter 2.3 cm). Five biomarkers were identified as being suitable for detection and identification of ATAAs using qRT-PCR analysis of whole blood. Analysis of 41 samples (19 small, 13 medium-sized, and 9 large ATAAs) demonstrated the overexpression of 3 of these transcript b...
American journal of physiology. Heart and circulatory physiology, 2000
Adenosine-enhanced ischemic preconditioning (APC) extends the protection afforded by ischemic pre... more Adenosine-enhanced ischemic preconditioning (APC) extends the protection afforded by ischemic preconditioning (IPC) by both significantly decreasing infarct size and significantly enhancing postischemic functional recovery. The purpose of this study was to determine whether APC is modulated by ATP-sensitive potassium (K(ATP)) channels and to determine whether this modulation occurs before ischemia or during reperfusion. The role of K(ATP) channels before ischemia (I), during reperfusion (R), or during ischemia and reperfusion (IR) was investigated using the nonspecific K(ATP) blocker glibenclamide (Glb), the mitochondrial (mito) K(ATP) channel blocker 5-hydroxydecanoate (5-HD), and the sarcolemmal (sarc) K(ATP) channel blocker HMR-1883 (HMR). Infarct size was significantly increased (P < 0.05) in APC hearts with Glb-I, Glb-R, and 5-HD-I treatment and partially with 5-HD-R. Glb-I and Glb-R treatment significantly decreased APC functional recovery (P < 0.05 vs. APC), whereas 5-H...
Previously, we have shown that potassium and magnesium (K-Mg, 20 mM each) cardioplegia ameliorate... more Previously, we have shown that potassium and magnesium (K-Mg, 20 mM each) cardioplegia ameliorated cytosolic calcium ([Ca2+]i) accumulation and was associated with enhanced functional recovery after surgically induced global ischemia in the aged heart. K-Mg cardioplegia was also shown to enhance cytosolic cytochrome oxidase I activity and mRNA levels, suggesting that enhanced functional recovery may involve the preservation of high-energy phosphates. To investigate this hypothesis, 31P nuclear magnetic resonance was used to measure serial alterations in phosphocreatine (PCr), inorganic phosphate, nucleoside triphosphate (NTP), intracellular free magnesium (Mgf), and intracellular pH (pHi) in Langendorff-perfused, aged (135 wk) rabbit hearts during preischemia, global ischemia (30 min), and reperfusion (30 min). K-Mg cardioplegia retarded PCr depletion (P < 0.05) and significantly enhanced NTP preservation (P < 0.05) during ischemia and reperfusion. K-Mg cardioplegia also atten...
Cytosolic calcium in the myocardium is rapidly accumulated during ischemia and has been correlate... more Cytosolic calcium in the myocardium is rapidly accumulated during ischemia and has been correlated with the attenuation of functional recovery in the myocardium. The aged myocardium is more sensitive to ischemia and accumulates significantly more cytosolic calcium than either the newborn or the mature myocardium. Modification of the age-related propensity to increased cytosolic calcium accumulation may be achieved through the use of magnesium or potassium/magnesium cardioplegia. Improved postischemic ventricular function obtained with magnesium or potassium/magnesium cardioplegia may have important implications in the reduction of myocardial morbidity and mortality.
Both heat-shock protein (HSP) 70 and its mRNA are induced in the ischemic myocardium. The inducto... more Both heat-shock protein (HSP) 70 and its mRNA are induced in the ischemic myocardium. The inductor stimuli are, however, not known. Stretch and ischemic metabolic alterations are the two most likely HSP70 inductors. To assess whether anaerobic metabolism induces HSP70 mRNA expression, 61 rat hearts were perfused with Krebs-Henseleit buffer (37 degrees C) in a modified Langendorff apparatus built for rapid switching between pressure-controlled and flow-controlled perfusions. Each heart was initially perfused for 30 minutes at a constant perfusion pressure of 65 mm Hg. Subsequently, separate hearts were perfused at a constant flow of 8, 6, 4, 2, 1, 0.5, or 0 mL/min for 30 minutes using a nonpulsatile pump (n = 5, each perfusion level). Hemodynamic measurements demonstrated a linear correlation between coronary flow and both perfusion pressure and developed pressure (r = .94 and r = .89, respectively; P < .0001 for both comparisons). Diastolic pressure at the end of the perfusions i...
We carried out a study to relate the effect of the type of dietary fat and ethanol on antioxidant... more We carried out a study to relate the effect of the type of dietary fat and ethanol on antioxidant enzyme mRNA levels in liver in the intragastric feeding rat model. Different types of dietary fat were administered [saturated fat (SE), corn oil (CE) and fish oil (FE)] with ethanol to induce varying severities of liver injury. Ethanol-fed rats were pair-fed with dextrose-fed controls that received isocaloric amounts of dextrose. All animals were killed at 1 month and the following studies were carried out: evaluation of severity of pathologic liver injury, mRNA quantitation for catalase, glutathione peroxidase (GPx), and manganese superoxide dismutase (MnSOD), microsomal conjugated dienes, and hydrogen peroxide. SE animals had no liver injury, FE animals had severe liver injury, and CE animals had moderate liver injury. Ethanol induced GPx mRNA in all dietary groups, with the highest levels seen in the FE group. The pattern of catalase mRNA induction was similar to that of GPx mRNA. I...
Background: We examined the effects of in vivo administration of a potent inhibitor of iron-depen... more Background: We examined the effects of in vivo administration of a potent inhibitor of iron-dependent lipid peroxidation, lazaroid U74389G, on lung preservation.
Previously described mitochondrial isolation methods using differential centrifugation and/or Fic... more Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 10(10) viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.
ABSTRACT The purpose of this study was to develop an isolated, pulsatile blood-perfused rat lung ... more ABSTRACT The purpose of this study was to develop an isolated, pulsatile blood-perfused rat lung model that allows us to evaluate the preserved lung functions. Lungs isolated from Sprague-Dawley rats, were perfused with venous whole blood by either a pulsatile or constant flow. The effuent was continuously deoxygenated with a 95% N2/5% CO2 gas mixture. Airway resistance, lung compliance, elastic work, flow resistive work, pulmonary vascular resistance, and blood gas analysis were assessed. Pressor responses toN G -monomethyl-l-arginine (l-NMMA) were compared between pulsatile and constant blood flow. At a flow of 0.1 ml/g body weight/min, pulsatile perfusion allowed for stable perfusion at least for 2h (mean 162.5±15.1 min) with stable aerodynamic and hemodynamic variables including blood gas tensions, whereas constant perfusion resulted in immediate lung failure. Whenl-NMMA was added to the perfusate, the mean pulmonary artery pressure did not show any change in the constant flow (6.0±2.6% increase), but did show a significant increase in the pulsatile flow (45±11% increase). Pulsatile blood flow reduced the pulmonary vascular resistance relative to the constant flow and allowed for a 2-h perfusion period to evaluate the lung function. The vasorelaxant mechanism in the pulsatile perfusion is related in part to the endothelial-dependent relaxation observed in the nitric oxide pathway.
1 other HighWire hosted article: This article has been cited by [PDF] [Full Text] [Abstract] , No... more 1 other HighWire hosted article: This article has been cited by [PDF] [Full Text] [Abstract] , November 1, 2010; 88 (2): 211-218. Cardiovasc Res M. Gucek and E. Murphy What can we learn about cardioprotection from the cardiac mitochondrial proteome?
The Journal of Thoracic and Cardiovascular Surgery, 1996
Objective: The effect of cardioplegic solutions with high concentrations of potassium or magnesiu... more Objective: The effect of cardioplegic solutions with high concentrations of potassium or magnesium (or both) on cytosolic calcium accumulation was investigated with fura-2 in isolated perfused mature (n = 24) and aged (n = 24) rabbit hearts. Methods: We compared cytosolic calcium accumulation before ischemia (control), during 30 minutes of ischemia and 30 minutes of reperfusion under global ischemia, or after treatment with potassium (20 mmol/L), magnesium (20 retool/L), or both. Results: Cytosolic calcium accumulation was increased during global ischemia in the mature heart (from 178.7 +-. 24.2 in the control group to 393.6 +-25.5 nmol/L; p < 0.005) and in the aged heart (from 187.4 + 18.7 in the control group to 501.0 +-46.1 nmol/L; p < 0.005). Potassium reduced cytosolic calcium accumulation during ischemia in both the mature and aged hearts (300.9 _ 23.2 and 365.2 -27.7 nmol/L, respectively; p < 0.05 vs global ischemia). Magnesium and potassium/magnesium completely controlled cytosolic calcium accumulation in the mature heart (198.7 +-27.5 nmol/L; p < 0.01 vs global ischemia andp < 0.05 vs potassium: 182.3 _+ 22.7 nmol/L;p < 0.05 vs global ischemia and potassium, respectively). Magnesium and potassium/magnesium attenuated cytosolic calcium accumulation in the aged heart (261.3 -+ 26.7, 262.3 +-. 25.2 nmol/L, respectively; p < 0.01 vs global ischemia). These changes in cytosolic calcium accumulation correlated with improved postischemic ventricular function. To investigate the mechanism(s) of magnesium-supplemented cardioplegic inhibition of cytosolic calcium accumulation, we performed parallel studies (n = 43) using nifedipine, ryanodine, and dimethylthiourea. Nifedipine with or without ryanodine reduced eytosolic calcium accumulation. Dimethylthiourea did not alter cytosolic calcium accumulation during global ischemia. Our results suggest that eytosolic calcium accumulation during global ischemia was mainly increased via the sarcolemmal 1-type calcium channel and the sarcoplasmic reticulum calcium-release channel. The modulating action of potassium/ magnesium cardioplegia on cytosolic calcium accumulation during ischemia would appear to act through the inhibition of the myocardial 1-type calcium channel and the sarcoplasmic reticulum calcium-release channel. Conclusion: Senescent cardiac dysfunction correlates with increased ischemia-induced cytosolic calcium accumulation. Magnesium-supplemented potassium cardioplegia ameliorates this age-related phenomenon at normothermia and may have important implications in myocardial protection in the elderly population. (J Thorac Cardiovasc Surg 1996;112:175-84) From the Division
We have established a highly sensitive functional screen for the isolation of cDNAs encoding cAMP... more We have established a highly sensitive functional screen for the isolation of cDNAs encoding cAMP phosphodiesterases (PDEs) by complementation of defects in a Saccharomyces cereuisiae strain lacking both endogenous cAMP PDE genes, PDEl and PDE2. Three groups of cDNAs corresponding to three distinct human genes encoding CAMP-specific PDEs were isolated from a human glioblastoma cDNA library using this functional screen. Two of these genes are closely related to the Drosophila dunce CAMP-specific PDE. The third gene, which we named HCP1, encoded a novel CAMP-specific PDE. HCPl has an amino acid sequence related to the sequences of the catalytic domains of all cyclic nucleotide PDEs. HCPl is a high affinity CAMPspecific PDE (K, = 0.2 pM) that does not share other properties of the CAMP-specific PDE family, i.e. extensive sequence homology to the Drosophila dunce cAMP PDE and sensitivity to rolipram and R020-1724. The PDE activity of HCPl is not sensitive to cGMP or other inhibitors of the cGMP-inhibitable PDEs, such as milrinone. The biochemical and pharmacological properties of HCPl suggest that it is a member of a previously undiscovered cyclic nucleotide PDE family. Northern blot analysis indicates that high levels of HCP 1 mRNA are present in human skeletal muscle.
Journal of Molecular and Cellular Cardiology, 1987
A system for RNA transcription in vitro was established in order to determine the relative rate o... more A system for RNA transcription in vitro was established in order to determine the relative rate of RNA synthesis in neonatal and adult rat myocardial cells. This assay system optimizes the incorporation of [3H]UMP into RNA by using 3.5 x 107 myocardial-cell nuclei, and minimizes RNA degradation for at least 1 h in transcription in vitro, by the addition of human placental RNAase inhibitor. A 1000 increase in the incorporation of [3H]UMP into myocardial-cell RNA was found on addition of this inhibitor. Myocardialcell nuclei derived from 5-, 10-, 15-, 20-, and > 100-day-old rat hearts indicated that there is a progressive decrease in RNA synthesis with age. A 3-fold increase in RNA synthesis in 5-day-old myocardial cell nuclei as compared with 20-day-old rat heart was found. RNA synthesis in the adult myocardial cell nuclei decreased more than 10-fold in comparison with the 5-day-old newborn. The incorporation of [3H]UMP into rat liver nuclear RNA was 3-fold greater than in the myocardial-cell nuclear RNA, even when compared with the highly active transcription of 12-day-old heart nuclei. In order to determine the relationship between total RNA synthesis and the extent of specific gene expression in myocardial-cell nuclei during development, two distinct cDNA probes were used for Northern-blot analysis. Our results indicate that myosin-heavy-chain gene expression is remarkably decreased with age, whereas the 'housekeeping' gene is continually expressed independently of age.
Uploads
Papers by James McCully