Original Article
Atrial natriuretic peptide property on the ischemic myocardium inducing HSP72
Asian Cardiovascular & Thoracic Annals 2014, Vol. 22(3) 301–308 ß The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0218492313484735 aan.sagepub.com
Silvio Olivito1, Massimo Chello2, Elvio Covino2 and Pasquale Mastroroberto1
Abstract Background: the purpose of this study was to investigate the effectiveness of atrial natriuretic peptide on ischemic myocardium through the induction of heat-shock protein 72. Methods: 30 isolated rabbit hearts perfused on isolated heart apparatus were randomly assigned to receive either warm Krebs-Henseleit solution with 1 mmol L1 atrial natriuretic peptide (n ¼ 15) or warm Krebs-Henseleit solution without atrial natriuretic peptide (n ¼ 15) in preischemic, ischemic, and postischemic conditions. In all rabbit hearts, global ischemia was produced by clamping the aortic and atrial inflow lines. Concentrations of atrial natriuretic peptide were measured in hearts with left ventricular dysfunction following ischemia, and correlated with the hypertrophic growth sustained by overexpression of heat-shock protein 72 microRNA-133. Results: the levels of atrial natriuretic peptide were markedly higher in the group that received atrial natriuretic peptide, and strongly correlated with both band lengths of heat-shock protein 72 and overexpression of microRNA-133 in the hypertrophic myocyte. Conclusions: perfusion levels of atrial natriuretic peptide induce increased expression of heat-shock protein 72 microRNA-133 in dysfunctional left ventricle.
Keywords Atrial natriuretic factor, HSP72 heat-shock proteins, Hemodynamics, Hyperthermia, induced, Myocardial reperfusion
Introduction The assessment of myocardial function is an important factor in the interpretation of the surgical outcomes. Left ventricular dysfunction can be classified in many ways such as symptomatic vs. asymptomatic, global vs. regional, reversible vs. permanent, systolic vs. diastolic, ischemic vs. nonischemic, or primary vs. secondary. Several methods are available to detect global or regional left ventricular dysfunction, of which, radionuclide ventriculography and echocardiography are most frequently adopted by clinical investigators. Nevertheless, radionuclide ventriculography presents some limitations in terms of availability, cost, and the need to administer a significant dose of radioactive material in experimental models, whereas echocardiography, although widely available and less expensive, is less objective and requires experienced personnel for the interpretation of data.1–3 Hence Chitwood and colleagues4 evaluated the applicability of pulse-transit
sonomicrometry to measure global ventricular function during cardiac surgery. Atrial natriuretic peptide (ANP) has been demonstrated to improve left ventricular systolic function and protect against reoxygenation-induced hypercontracture in isolated cardiomyocytes by increasing cyclic guanosine monophosphate synthesis.5 Amrani and colleagues6,7 documented a protective effect of heat stress on both endothelial and mechanical function after cardioplegic arrest. Other studies have shown that 1 Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Italy 2 Integrated Research Center, Campus Bio-Medico University of Rome, Italy
Corresponding author: Pasquale Mastroroberto, MD, Department of Experimental and Clinical Medicine, Magna Graecia University, Viale Europa, 88100 Catanzaro, Italy. Email: [email protected]
Downloaded from aan.sagepub.com at FLORIDA INTERNATIONAL UNIV on May 27, 2015
302
Asian Cardiovascular & Thoracic Annals 22(3)
heat-shock proteins (HSP) can be released rapidly in the heart after hyperthermic conditions.8–11 Because transcription factors and multiple cellular signaling molecules are a decisive factor in the regulation of muscle proliferation and differentiation,12 we considered that it was important to assess microRNA-133 (miR-133) levels in the regulation of HSP72 expression. Cardiac myocytes proliferate rapidly during embryogenesis, but adult cardiac myocytes lose their proliferative capacity and respond to mechanical and pathological stimuli by hypertrophic growth, and the reactivation of cardiac fetal genes in postnatal cardiomyocytes can regulate hypertrophic cardiac growth and heart regeneration.13 The aims of our study were to determine whether ANP could cause overexpression of miR-133 by activation of heat-shock protein 72 (HSP72) in left ventricular dysfunction in hypertrophic myocytes, and to assess the effect of ANP on infarct size in a model of rabbit coronary occlusion.
Materials and methods The hearts from 30 New Zealand White rabbits (1200– 1500 g) were perfused on Isolated Heart Size 5 Type 843 apparatus (Hugo Sachs Elektronik-Harvard Apparatus GmbH March-Hugstetten Germany) and assigned to two groups, using a simple numerical random allocation. Body weights, heart weights, and the heart/body weight ratio in the 2 groups are given in Table 1. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals according to the guidelines of US National Institutes of Health. The rabbits were anesthetized by continuous intravenous infusion of ketamine hydrochloride 10 mg kg1 h1 and xylazine 2 mg kg1 h1, and paralysis was obtained using pancuronium bromide 0.3 mg kg1 h1. An endotracheal tube was inserted through a tracheotomy, and pressure-controlled ventilation (Servo 300; Siemens, Solna, Sweden) and the fraction of inspired oxygen were maintained. Positive end-expiratory pressure, peak inspiratory pressure, mean airway pressure, and tidal volume were measured using ventilator transducers. Group 1 consisted of 15 isolated rabbit hearts that received warm KrebsHenseleit buffer with 1 mmol L1ANP during the
Table 1. Isolated heart data. Variable
Group 1
Group 2
Body weight (g) Heart weight (mg) Heart weight/body weight (mg g1)
1347 68 1221 25 0.90 0.14
1230 85 1157 9 0.94 0.56
periods of pre-ischemia, ischemia, and post-ischemia; a control group of 15 rabbit hearts received KrebsHenseleit buffer without ANP (group 2). In all rabbits, sodium heparin at dose of 200 IU per 100 g body weight was administered 20 min prior to sacrifice. The heart was rapidly excised and immediately mounted on Isolated Heart Size 5 Type 843 apparatus (for hearts of rabbits up to 2.5 kg body weight) in 2 operating modes (working heart and Langerdoff heart), using medetomidine and xylazine or fentanyl. The isolated heart was perfused at a constant pressure of 80 mm Hg with Krebs-Henseleit buffer for a 10-min period of stabilization. The temperature of this solution was maintained at 37 C by a thermostatic circuit. Two different principles of flow measurement were used (electromagnetic and ultrasonic), maintaining the flow rate at 22–25 mL min1. Cardiac output was calculated as the combined aortic and coronary flow. The coronary flow was measured using a D-79232 electromagnetic flowmeter (Hugo Sachs Elektronik, March-Hugstetten, Germany). Data of aortic and coronary flow were recorded and compared between the two groups. Isovolumetric measurement of the internal cardiac pressure was performed with a latex balloon passed into the left ventricle by means of a steel catheter, and calculated as the difference between the systolic and diastolic pressures with the transducer amplifier module of the apparatus. Basic rhythm and stimulus width as well as the stimulus amplitude were adjusted with an electrical square-wave stimulator (P Type 201; Hugo Sachs Elektronik-Harvard Apparatus GmbH, March-Hugstetten Germany). After an initial washout period, the hearts were perfused in the working heart mode for 25 min while measuring heart rate, coronary flow, and cardiac output every 8 min, and the averages were considered as preischemic indices. After 10 min of Langendorff perfusion with a medium flow rate of 70 mL min1, the hearts were subjected to global ischemia by clamping the aortic and atrial inflow lines of the apparatus, and maintained at 37.0 C for 15 min in the heart chamber with a thermostatic circuit (ischemic conditions). The hearts were reperfused for 15 min in the Langendorff mode, followed by perfusion in the working mode for the next 15 min while evaluating postischemic cardiac function. The coronary effluent was collected for 5 min immediately after stabilization (baseline) and at 10 and 30 min in the postischemic period for lactate dehydrogenase (LDH) and creatine kinase (CK-MB) estimations in both groups. The values of LDH and CK-MB were compared with the degree of left ventricular dysfunction. A specimen was immediately excised from the apex of the left ventricle to evaluate infarct size. A second and third specimens were obtained during the ischemic period and 15 min after
Downloaded from aan.sagepub.com at FLORIDA INTERNATIONAL UNIV on May 27, 2015
Olivito et al.
303
ischemia. The specimens were immediately frozen in liquid nitrogen and stored until examination. After completion of all measurements, the hearts were arrested in diastole using 20 mL of 20% potassium chloride solution. Isolated rabbit hearts presenting with heart rates
Atrial natriuretic peptide property on the ischemic myocardium inducing HSP72
Asian Cardiovascular & Thoracic Annals 2014, Vol. 22(3) 301–308 ß The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0218492313484735 aan.sagepub.com
Silvio Olivito1, Massimo Chello2, Elvio Covino2 and Pasquale Mastroroberto1
Abstract Background: the purpose of this study was to investigate the effectiveness of atrial natriuretic peptide on ischemic myocardium through the induction of heat-shock protein 72. Methods: 30 isolated rabbit hearts perfused on isolated heart apparatus were randomly assigned to receive either warm Krebs-Henseleit solution with 1 mmol L1 atrial natriuretic peptide (n ¼ 15) or warm Krebs-Henseleit solution without atrial natriuretic peptide (n ¼ 15) in preischemic, ischemic, and postischemic conditions. In all rabbit hearts, global ischemia was produced by clamping the aortic and atrial inflow lines. Concentrations of atrial natriuretic peptide were measured in hearts with left ventricular dysfunction following ischemia, and correlated with the hypertrophic growth sustained by overexpression of heat-shock protein 72 microRNA-133. Results: the levels of atrial natriuretic peptide were markedly higher in the group that received atrial natriuretic peptide, and strongly correlated with both band lengths of heat-shock protein 72 and overexpression of microRNA-133 in the hypertrophic myocyte. Conclusions: perfusion levels of atrial natriuretic peptide induce increased expression of heat-shock protein 72 microRNA-133 in dysfunctional left ventricle.
Keywords Atrial natriuretic factor, HSP72 heat-shock proteins, Hemodynamics, Hyperthermia, induced, Myocardial reperfusion
Introduction The assessment of myocardial function is an important factor in the interpretation of the surgical outcomes. Left ventricular dysfunction can be classified in many ways such as symptomatic vs. asymptomatic, global vs. regional, reversible vs. permanent, systolic vs. diastolic, ischemic vs. nonischemic, or primary vs. secondary. Several methods are available to detect global or regional left ventricular dysfunction, of which, radionuclide ventriculography and echocardiography are most frequently adopted by clinical investigators. Nevertheless, radionuclide ventriculography presents some limitations in terms of availability, cost, and the need to administer a significant dose of radioactive material in experimental models, whereas echocardiography, although widely available and less expensive, is less objective and requires experienced personnel for the interpretation of data.1–3 Hence Chitwood and colleagues4 evaluated the applicability of pulse-transit
sonomicrometry to measure global ventricular function during cardiac surgery. Atrial natriuretic peptide (ANP) has been demonstrated to improve left ventricular systolic function and protect against reoxygenation-induced hypercontracture in isolated cardiomyocytes by increasing cyclic guanosine monophosphate synthesis.5 Amrani and colleagues6,7 documented a protective effect of heat stress on both endothelial and mechanical function after cardioplegic arrest. Other studies have shown that 1 Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Italy 2 Integrated Research Center, Campus Bio-Medico University of Rome, Italy
Corresponding author: Pasquale Mastroroberto, MD, Department of Experimental and Clinical Medicine, Magna Graecia University, Viale Europa, 88100 Catanzaro, Italy. Email: [email protected]
Downloaded from aan.sagepub.com at FLORIDA INTERNATIONAL UNIV on May 27, 2015
302
Asian Cardiovascular & Thoracic Annals 22(3)
heat-shock proteins (HSP) can be released rapidly in the heart after hyperthermic conditions.8–11 Because transcription factors and multiple cellular signaling molecules are a decisive factor in the regulation of muscle proliferation and differentiation,12 we considered that it was important to assess microRNA-133 (miR-133) levels in the regulation of HSP72 expression. Cardiac myocytes proliferate rapidly during embryogenesis, but adult cardiac myocytes lose their proliferative capacity and respond to mechanical and pathological stimuli by hypertrophic growth, and the reactivation of cardiac fetal genes in postnatal cardiomyocytes can regulate hypertrophic cardiac growth and heart regeneration.13 The aims of our study were to determine whether ANP could cause overexpression of miR-133 by activation of heat-shock protein 72 (HSP72) in left ventricular dysfunction in hypertrophic myocytes, and to assess the effect of ANP on infarct size in a model of rabbit coronary occlusion.
Materials and methods The hearts from 30 New Zealand White rabbits (1200– 1500 g) were perfused on Isolated Heart Size 5 Type 843 apparatus (Hugo Sachs Elektronik-Harvard Apparatus GmbH March-Hugstetten Germany) and assigned to two groups, using a simple numerical random allocation. Body weights, heart weights, and the heart/body weight ratio in the 2 groups are given in Table 1. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals according to the guidelines of US National Institutes of Health. The rabbits were anesthetized by continuous intravenous infusion of ketamine hydrochloride 10 mg kg1 h1 and xylazine 2 mg kg1 h1, and paralysis was obtained using pancuronium bromide 0.3 mg kg1 h1. An endotracheal tube was inserted through a tracheotomy, and pressure-controlled ventilation (Servo 300; Siemens, Solna, Sweden) and the fraction of inspired oxygen were maintained. Positive end-expiratory pressure, peak inspiratory pressure, mean airway pressure, and tidal volume were measured using ventilator transducers. Group 1 consisted of 15 isolated rabbit hearts that received warm KrebsHenseleit buffer with 1 mmol L1ANP during the
Table 1. Isolated heart data. Variable
Group 1
Group 2
Body weight (g) Heart weight (mg) Heart weight/body weight (mg g1)
1347 68 1221 25 0.90 0.14
1230 85 1157 9 0.94 0.56
periods of pre-ischemia, ischemia, and post-ischemia; a control group of 15 rabbit hearts received KrebsHenseleit buffer without ANP (group 2). In all rabbits, sodium heparin at dose of 200 IU per 100 g body weight was administered 20 min prior to sacrifice. The heart was rapidly excised and immediately mounted on Isolated Heart Size 5 Type 843 apparatus (for hearts of rabbits up to 2.5 kg body weight) in 2 operating modes (working heart and Langerdoff heart), using medetomidine and xylazine or fentanyl. The isolated heart was perfused at a constant pressure of 80 mm Hg with Krebs-Henseleit buffer for a 10-min period of stabilization. The temperature of this solution was maintained at 37 C by a thermostatic circuit. Two different principles of flow measurement were used (electromagnetic and ultrasonic), maintaining the flow rate at 22–25 mL min1. Cardiac output was calculated as the combined aortic and coronary flow. The coronary flow was measured using a D-79232 electromagnetic flowmeter (Hugo Sachs Elektronik, March-Hugstetten, Germany). Data of aortic and coronary flow were recorded and compared between the two groups. Isovolumetric measurement of the internal cardiac pressure was performed with a latex balloon passed into the left ventricle by means of a steel catheter, and calculated as the difference between the systolic and diastolic pressures with the transducer amplifier module of the apparatus. Basic rhythm and stimulus width as well as the stimulus amplitude were adjusted with an electrical square-wave stimulator (P Type 201; Hugo Sachs Elektronik-Harvard Apparatus GmbH, March-Hugstetten Germany). After an initial washout period, the hearts were perfused in the working heart mode for 25 min while measuring heart rate, coronary flow, and cardiac output every 8 min, and the averages were considered as preischemic indices. After 10 min of Langendorff perfusion with a medium flow rate of 70 mL min1, the hearts were subjected to global ischemia by clamping the aortic and atrial inflow lines of the apparatus, and maintained at 37.0 C for 15 min in the heart chamber with a thermostatic circuit (ischemic conditions). The hearts were reperfused for 15 min in the Langendorff mode, followed by perfusion in the working mode for the next 15 min while evaluating postischemic cardiac function. The coronary effluent was collected for 5 min immediately after stabilization (baseline) and at 10 and 30 min in the postischemic period for lactate dehydrogenase (LDH) and creatine kinase (CK-MB) estimations in both groups. The values of LDH and CK-MB were compared with the degree of left ventricular dysfunction. A specimen was immediately excised from the apex of the left ventricle to evaluate infarct size. A second and third specimens were obtained during the ischemic period and 15 min after
Downloaded from aan.sagepub.com at FLORIDA INTERNATIONAL UNIV on May 27, 2015
Olivito et al.
303
ischemia. The specimens were immediately frozen in liquid nitrogen and stored until examination. After completion of all measurements, the hearts were arrested in diastole using 20 mL of 20% potassium chloride solution. Isolated rabbit hearts presenting with heart rates
