| BackgroundExperimental evidence has provided strong supports for the cardioprotective effects of"Glucose-insulin-potassium (GIK)"solution since its introduction as an adjunct to the contemporary management of acute myocardial ischemia (AMI). GIK solution, however, has not been uniformly successful in clinical practice over the last few decades and few trials and experiments to date have been adequately powered to explore the mechanism underlying the mixed results. Although it has been proposed that insulin therapy alone without achieving euglycemia may not improve outcomes in AMI, it remains completely unevidenced whether insulin-titrated euglycemia maintenance is requisite for the cardioprotective effects of insulin. The definite relationship between glycemic control, cardioprotection, and insulin therapy itself requires further elucidation.Meanwhile, our recent studies as well as others'have proposed direct cell survival signalings favorably modulated by insulin against myocardial ischemia/reperfusion (MI/R) injury. In addition to its anti-apoptotic action, anti-inflammatory, anti-oxidative stress and positive inotropic effects have also been well documented, and are believed to further enhance cardiac performanc after reperfusion. However, latest reports from the research on critically ill patients and animal models suggested that the survival benefits from intensive insulin therapy were due to successful glycemic control, rather than other glycemia-independent effects of insulin. However, cardiac outcome was not addressed, thus it remains unclear whether the well-documented benefits of insulin in the ischemic/reperfused heart should be attributed exclusively to glucose normalization, or combined with other mechanisms inherently originating from insulin per se. The clarification of the existence and contribution of glycemic-control-independent actions of insulin may greatly advance the understanding and application of GIK in AMI management.ObjectivesThe present study was designed to investigate, in a clinically relevant large animal model of myocardial ischemia/reperfusion (MI/R), the relationship between glycemia-dependent and -independent effects of insulin and their contributions to insulin's cardioprotection.Methods1. Endogenous insulin production in canines was abolished by peripancreatic vessel ligation. Aninals were then randomly assigned to four groups: Normal plasma insulin/euglycemia (NI/NG), normal insulin/hyperglycemia (NI/HG), high insulin/euglycemia (HI/NG), and high insulin/hyperglycemia (HI/HG), which were achieved by controlled intravenous infusion of glucose/insulin.2. Anesthetized open-chest dogs were subjected to MI/R (50 min/4 h) by partially occluding the left anterior descending coronary artery (LAD) (80% reduction in its blood flow). Gluose/insulin administration started at 5 min before reperfusion and continued thoughout the experiment.3. Blood glucose and plasma concentrations of insulin, C-peptide and free fatty acid (FFA) were measured for metabolic control and evaluations. Blood samples from the left femoral artery and the anterior interventricular vein were analyzed for PO2, PCO2, pH, hemoglobin (Hb) concentration and oxygen saturation (SO2) in order to calculate oxygen consumption rate (MVO2) and respiratory quotient (RQ) of the LAD perfused region.4. Left ventricular (LV) pressure was continuously monitored by a hemodynamic analyzing system. Myocardial infarction was measured using the Evans blue/2,3,5-triphenyltetrazolium chloride (TTC) double staining. Plasma level of cardiac troponin-T (cTnT) was assayed as a biomarker for myocardial ischemic injury. Cell apoptosis was analyzed by immunohistochemical detection of caspase-3 activation and TUNEL assay.5. Myocardial inflammation was evaluated by histochemical detection of leukocyte infiltration as well as measurement of tissue myeloperoxidase (MPO) activity. Oxidative stress was estimated by measuring the tissue content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD).6. Strict criteria were established to guarantee that all animals included in the final data analysis were healthy and exposed to comparable degrees of regional myocardial ischemia.Results1. Canine acute MI/R model was successfully established. Significant myocardial infarction, cell apoptosis and necrosis, inflammatory reaction and oxidative stress were observed as expected.2. The ischemic insult due to pancreas surgery resulted in limited pancreatic inflammation and minimal influence on systemic inflammation and cardiac function within 5 hours'protocol. Plasma levels of amylase, lipase, cTnT and creatine kinase-MB, as well as hemodynamic indices at baseline and 5 hours after the pancreas surgery showed no statistical differences between pancreatic-ligated versus non-operated dogs subjected to MI/R.3. Hyperglycemia alone significantly aggravated MI/R injury, as evidenced by worse functional recovery, larger infarct size, increased cell apoptosis and necrosis, and enhanced inflammation and oxidative injury in HG groups compared to NG groups.4. Insulin increased conoray perfusion and metabolic efficiency as well as promoted contractile function of the I/R myocardium, which were minimally affected by glucose levels. Compared to NI groups, both HI groups showed higher rate-pressure product and +LVdp/dtmax with increased LAD blood flow during reperfusion. In addition, a robust elevation in anterior LV glucose uptake was also observed following insulin infusion, accompanied by a slight decline in FFA uptake and increase in respiratory quotient, suggesting insulin-mediatd switch of myocardial substrate uptake from fat to carbohydrate. Interestingly, MVO2 of the anterior LV was not elevated in HI groups though contractile function increased, indicating an apparent increase in O2 utilization efficiency.5. When blood glucose was clamped at physiologic level, insulin elevation (HI/NG vs. NI/NG) further exerted protection against MI/R injury, as evidenced by improved functional recovery, decreased myocardial infarct size, reduced cell necrosis and apoptosis, and alleviated inflammatory and oxidative stress. However, maintaining high glucose concentration in high insulin animals (HI/HG) markedly blunted or abolished the above protective effects of insulin.Conclusions1. Insulin-titrated maintenance of euglycemia protects heart from MI/R injury, whereas hyperglycemia oppositely affects MI/R injury and masks the insulin-induced cardiac benefits. Thus insulin-mediated hyperglycemia prevention is of critical importance to ensure insulin's full efficacy in protecting heart against MI/R.2. Insulin has direct cardioprotective effects independent of its glucose-lowering ability. Thus the cardiac benefits of insulin against I/R injury should not be attributed exclusively to glucose normalization.Part IIBackgroundMyocardial ischemia involves a large and progressive release of catecholamines from adrenergic nerve terminals, and overshooting of myocardialβ- adrenergic receptors (?-AR) by catecholamines may further aggravate MI/R injury, induce lethal arrhythmias, and contribute to post-infarction remodeling and heart failure.Increasing evidence showed that insulin, apart from its role in the metabolic regulation, participates in a variety of physiological and pathological cardiovascular process. In the above Part I study, we have demonstrated that insulin has direct cardioprotective effects independent of its glucose-lowering ability. For example, through the activation of a cell survival signaling"PI3K-Akt-eNOS", insulin exerts protection for the I/R heart by inhibiting cell apoptosis, preserving endothelial function, and suppressing myocardial inflammation and oxidative stress. Moreover, a direct positive inotropic effect of insulin has been reported as evidenced by increased contractile function in in vivo models and elevated twitch amplitude and calcium transient in single ventricular myocyetes.Previous investigations have revealed a counter-regulatory effect of insulin onβ-adrenergic catecholamine action in isolated human fat cells and cultured smooth muscle cells. Recent studies also provided important evidence for the existence of interaction between myocardial insulin signaling and ?-AR signaling. However, existing data concerning the functional effects of insulin on the myocardial response to ?-AR activation are quite limited and inconsistent. Moreover, the major cardiac effects of catecholamines are mediated by ?-AR through the activation of cAMP-dependent protein kinase A (PKA), which in turn phosphorylates a series of target moleculars responsible for the regulation of excitation-contraction coupling. Phospholamban is one of the PKA target proteins, and at the same time a major regulator of SERCA2, which modulates the rate of SR Ca2+ uptake, leads to the regulation of relaxation velocity, SR Ca2+ load and myocardial contractility. Several events associated with MI/R, such as catecholamines release, intracellular Ca2+ overload and oxidative stress, participate in the modulation of PLB phosphorylation and SERCA2 activity. However, it remains unclear howβ-AR stimulation influences these SR Ca2+ handling proteins in the I/R heart and whether these influences could be modified by the application of insulin.ObjectivesThis study aimed to investigate the ability of insulin to modulateβ-adrenergic actions on post-ischemic injury and myocardial contractility in acute MI/R and the underlying mechanism.Methods1. Isolated hearts from adult SD rats were subjected to MI/R (30 min/2 h). Hearts (n=16-20 per group) were randomized to receive one of the following treatments: (1) sham MI/R; (2) MI/R vehicle; (3) MI/R receiving insulin (10-7 mol/L) 5 min before and throughout reperfusion; (4) MI/R receiving ISO (10-9 mol/L) 10 min before ischemia and throughout reperfusion; (5) MI/R receiving insulin 5 min before reperfusion plus ISO (10-9 mol/L) 10 min before ischemia, both throughout reperfusion.2. A water-filled latex balloon was inserted into the left ventricle; cardiac function was continuously recorded by a hemodynamic analyzing system via a pressure transducer connected to the balloon. Myocardial infarction was measured using the Evans blue/TTC double staining. Coronary effluent was collected and creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured spectrophotometrically. Cardiomyocyte apoptosis was analyzed by detection of DNA fragmentation (DNA ladders) and TUNEL in situ assay.3. Ca2+-ATPase activity of the isolated SR membrane was measured in the reperfused hearts. Expressions of SERCA2a, PLB and phosphorylated PLB in the SR were determined by Western blot. Tissue homogenates were prepared for non-radioactive determination of PKA activity using PepTag Assay.4. Calcium-tolerant ventricular myocytes were isolated from adult male rats by a standard enzymatic technique. Isolated myocytes were exposed to simulated ischemia for 15 min using chemical anoxia solution, and then reperfused with one of the following agents for another 30 min: (1) vehicle; (2) insulin (10-8 -10-5 mol/L); (3) isoproterenol (ISO, 10-9 mol/L); (4) ISO (10-9 mol/L) +insulin (10-8-10-5 mol/L). The mechanical contraction and Ca2+ transients of ventricular myocytes were assessed by a video-based motion edge-detection system. Responses to the above agents were also observed in normal myocytes.Results1. ISO administration produced a transient increase in HR and LVSP, followed by a sharp decline after 1 h reperfusion and end up with a greatly dampened HR and LVDP and elevated LVEDP compared with the untreated I/R hearts, suggesting thatβ-AR activation during MI/R aggravates post-ischemic cardiac dysfunction. More importantly, insulin largely reversed the ISO-induced functional impairment, inhibiting ISO-induced declines in HR and LVSP by 34.0% and 23.0% respectively, and preventing ISO-induced elevation in LVEDP by 28.7% (n=8, all P<0.05).2. ISO alone resulted in enlarged infarct size, elevated CK and LDH activity and increased apoptotic index in I/R hearts compared with vehicle, which were all inhibited by treatment of insulin (n=8, all P<0.05).3. In normal ventricular myocytes, insulin (10-7 mol/L) exerted significant inotropic effect as evidenced by increased peak twitch amplitude (PTA) and calcium transient amplitude (ΔFFI). ISO with a submaximal concentration of 10-9 mol/L, showed an even greater inotropic effect with a 19.1% increase of PTA and 85% increase ofΔFFI. Exposure to insulin (10-8-10-6 mol/L), either before or after the administration of ISO, displayed no significant differences with ISO alone.4. In simulated I/R cardiomyocytes, insulin (10-7mol/L) and ISO(10-9 mol/L)both mediated distinct positive inotropic responses, with 17% and 81% increases of PTA along with 15% and 109% increases ofΔFFI, respectively. When applied together, however, insulin attenuated the inotropic response to ISO as evidenced by an 18.7% reduction in PTA and a 23.9% reduction inΔFFI (n=20 myocytes from 8 hearts, both P<0.05). This inhibitory effect also acts in a concentration-dependent manner (10-8-10-5 mol/L).5. Both protein expression and activity of SERCA2a in untreated I/R hearts were significantly reduced compared with sham hearts, and were further depressed when ISO was applied. Reperfusion with insulin produced a 13.9% increase in SERCA2a activity compared with vehicle, and a 15.2% increase compared with ISO alone (both P<0.05). Phosphorylation of PLB at Ser16 was significantly decresed in I/R hearts. Insulin and ISO triggered a 4.2- and 3.1-fold increase in PLB phosphorylation, and insulin further facilitated ISO-induced PLB phosphorylation, a 1.7-fold increase relative to ISO alone (P<0.01). As for PKA activity, MI/R resulted in 2.5-fold increase in PKA activation compared with sham group. Application of ISO produced an overt elevation in PKA activity as evidenced by a 30.7-fold increase (P<0.01 vs. MI/R group). Interestingly, insulin itself exerted no significant influence on PKA activity, but markedly inhibited ISO-mediated PKA activation in I/R hearts (P<0.01). 1. Insulin significantly suppresses ISO-elicited cardiac injury including myocardial contractile dysfunction, cell apoptosis and necrosis in isolated I/R hearts, which may contribute to insulin-induced cardioprotection in MI/R.2. Insulin attenuates ISO-induced positive inotropic effect on simulated I/R cardiomyocytes in a concentration-dependent manner.3. The regulation of ?-adrenergic postreceptorial signaling such as PKA and its downstream calcium handling proteins is a likely mechanism that accounted for the insulin-induced counterbalance of -adrenergic activation in MI/R. Conclusions...
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