Novel insights into the cardio-protective effects of FGF21 in lean and obese rat hearts

Aims: Fibroblast growth factor 21 (FGF21) is a hepatic metabolic regulator with pleotropic actions. Its plasma concentrations are increased in obesity and diabetes; states associated with an increased incidence of cardiovascular disease. We therefore investigated the direct effect of FGF21 on cardio-protection in obese and lean hearts in response to ischemia. Methods and Results: FGF21, FGF21-receptor 1 (FGFR1) and beta-Klotho (βKlotho) were expressed in rodent, human hearts and primary rat cardiomyocytes. Cardiac FGF21 was expressed and secreted (real time RT-PCR/western blot and ELISA) in an autocrine-paracrine manner, in response to obesity and hypoxia, involving FGFR1-βKlotho components. Cardiac-FGF21 expression and secretion were increased in response to global ischemia. In contrast βKlotho was reduced in obese hearts. In isolated adult rat cardiomyocytes, FGF21 activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt), ERK1/2(extracellular signal-regulated kinase) and AMPK (AMP-activated protein kinase) pathways. In Langendorff perfused rat [adult male wild-type wistar] hearts, FGF21 administration induced significant cardio-protection and restoration of function following global ischemia. Inhibition of PI3K/Akt, AMPK, ERK1/2 and ROR-α (retinoic-acid receptor alpha) pathway led to significant decrease of FGF21 induced cardio-protection and restoration of cardiac function in response to global ischemia. More importantly, this cardio-protective response induced by FGF21 was reduced in obesity, although the cardiac expression profiles and circulating FGF21 levels were increased. Conclusion: In an ex vivo Langendorff system, we show that FGF21 induced cardiac protection and restoration of cardiac function involving autocrine-paracrine pathways, with reduced effect in obesity. Collectively, our findings provide novel insights into FGF21-induced cardiac effects in obesity and ischemia

Insulin inhibits cardiac contractility by inducing a Gi-biased β2-adrenergic signaling in hearts.

Insulin and adrenergic stimulation are two divergent regulatory systems that may interact under certain pathophysiological circumstances. Here, we characterized a complex consisting of insulin receptor (IR) and β2-adrenergic receptor (β2AR) in the heart. The IR/β2AR complex undergoes dynamic dissociation under diverse conditions such as Langendorff perfusions of hearts with insulin or after euglycemic-hyperinsulinemic clamps in vivo. Activation of IR with insulin induces protein kinase A (PKA) and G-protein receptor kinase 2 (GRK2) phosphorylation of the β2AR, which promotes β2AR coupling to the inhibitory G-protein, Gi. The insulin-induced phosphorylation of β2AR is dependent on IRS1 and IRS2. After insulin pretreatment, the activated β2AR-Gi signaling effectively attenuates cAMP/PKA activity after β-adrenergic stimulation in cardiomyocytes and consequently inhibits PKA phosphorylation of phospholamban and contractile responses in myocytes in vitro and in Langendorff perfused hearts. These data indicate that increased IR signaling, as occurs in hyperinsulinemic states, may directly impair βAR-regulated cardiac contractility. This β2AR-dependent IR and βAR signaling cross-talk offers a molecular basis for the broad interaction between these signaling cascades in the heart and other tissues or organs that may contribute to the pathophysiology of metabolic and cardiovascular dysfunction in insulin-resistant states

Do K_ATP channels open as a prominent and early feature during ischaemia in the Langendorff-perfused rat heart?

The objective was to investigate whether myocardial adenosine triphosphate-sensitive K<sup>+</sup> (K<sub>ATP</sub>) channels open during the first 10 min of regional ischaemia in Langendorff-perfused rat hearts. Changes in monophasic action potentials and arrhythmias were studied during myocardial ischaemia in both the presence and absence of pharmacological K<sub>ATP</sub> modulation. Ligation of the left main coronary artery for 10 min did not shorten the action potential duration (APD). The APD<sub>50</sub> and APD<sub>80</sub> (15.5 +/- 1.0 and 38.1 +/- 2.3 ms, respectively [mean +/- S.E., n = 15 hearts], immediately prior to ligation) increased transiently during the first 4 min of ligation (by 160 and 79% respectively, P < 0.05), before returning to pre-ligation values, but without a significant below-baseline-shortening. The cardiac electrogram showed no accompanying ventricular tachyarrhythmia (VT). These results raised the possibility that the myocardial K<sub>ATP</sub> channels had not opened during the ligation. The K<sub>ATP</sub> opener Ro 31-6930 (0.5 and 5 microM) shortened the APD50 and APD80 during coronary ligation, to significantly below both their control and pre-occlusion values (P < 0.05), and caused a concentration-dependent increase in both the incidence and duration of VT during the ligation. Ro 31-6930 at 5 microM also shortened APD50 and APD80 even before ligation (by 50 and 62% respectively, P < 0.05), and abolished the normal APD-lengthening seen during ischaemia. The K<sub>ATP</sub> blocker glibenclamide (1 μM) abolished both the APD-shortening and pro-arrhythmic effects of the K<sub>ATP</sub> opener, both before and during coronary ligation, yet when delivered on its own, at the same concentration which abolished the effects of K<sub>ATP</sub> activation, it had no significant effect on the APD changes seen during the coronary ligation alone. These results suggest that, in Langendorff-perfused rat hearts in the absence of drugs, K<sub>ATP</sub> channels do not open during early myocardial ischaemia

ATP-sensitive potassium channel subcellular trafficking during ischemia, reperfusion, and preconditioning

Ischemic preconditioning is an endogenous cardioprotective mechanism in which short periods of ischemia and reperfusion provide protection when given before a subsequent ischemic event. Early mechanistic studies showed ATP-sensitive potassium (KATP) channels to play an important role in ischemic preconditioning. KATP channels link intracellular energy metabolism to membrane excitability and contractility. It is thought that KATP channels provide a cardioprotective role during ischemia by inducing action potential shortening, reducing an excessive Ca^2+ influx, and by preventing arrhythmias. However, the mechanisms by which KATP channels protect during ischemic preconditioning are not known. In this study, we investigated a novel potential mechanism in which alterations in subcellular KATP channel trafficking during ischemia and ischemic preconditioning may result in altered levels of surface channel density, and therefore, a greater degree of cardioprotection. In the optimization of our experiments, we compared various antibodies for their specificity and sensitivity for channel subunit detection in immunoblotting. In addition, we examined the effects of varying salt concentrations during tissue homogenization in order to determine the optimal conditions for protein isolation. Furthermore, we examined the effect of heating the samples prior to SDS-PAGE for improved detection of channel proteins by immunoblotting. The subcellular trafficking of some membrane proteins is altered by ischemia. For example, the glucose transporter, Glut4, translocates from endosomal compartments to the sarcolemma (Sun, Nguyen, DeGrado, Schwaiger, & Brosius, 1994). Conflicting data exists regarding the effects of ischemia on KATP channel subcellular trafficking and the regulation of KATP channel surface density (Edwards et al., 2009 and Bao, Hadjiolova, Coetzee, & Rindler, 2011). We therefore, sought to test our hypothesis that KATP channels are internalized from the surface of cardiomyocytes to endosomal compartments during ischemia, and this internalization can be reduced and/or reversed by ischemic preconditioning. We subjected isolated Langendorff-perfused mouse hearts to ischemia, reperfusion, or ischemic preconditioning events and measured the density of KATP channels in the sarcolemmal and endosomal compartments. We also determined the degree of injury by staining heart slices with triphenyltetrazolium chloride and compared infarct sizes between hearts subjected to ischemia and ischemic preconditioning. Our data demonstrated that KATP channels are, in fact, internalized during ischemia and that reperfusion led to a slow recovery of surface KATP channel density. Interestingly, ischemic preconditioning reduced the size of infarcts induced by ischemia and also prevented the ischemia-induced decrease of KATP channel surface density, thereby, contributing to cardioprotection

The Lactate/Pyruvate Ratio of Metabolic Modulation Using Glucose Insulin Kalium and Lactate Solution and Their Effect on Functional Mechanical Recovery of the Isolated Perfused Heart

Metabolic modulation with Glucose Insulin Kalium (GIK) solution has beenreally well known in their capacity to improve post ischemic heart function. In this regardGIK intervention on post operative Coronary Artery Bypass Graft (CABG) can improveheart function recovery on reperfusion period (Goldhaber dan Weiss, 1992; Atwell et al.,1997). Post operative CABG intervention with GIK will produce a beneficial effect onthe elevation of heart energy to prevent ionic homeostasis disturbance and reactiveoxygen species (ROS) production that become the basis of reperfusion injury (Silvermandan Stern, 1994; Cross et al., 1995; Taegtmeyer et al., 1997; Opie, 1999; Lazar, 2002;Doenst et al., 2003; Trence et al., 2003).Many efforts have been made to clarify how exactly GIK works to improve postischemic heart function as in CABG. This is crucially done in order to be able to modifythe solution concerned. Although this solution has been clearly proved to improve postischemic heart function, it is not totally free from its adverse effect. Its main side effect isthat it can provoke hyperglycemic state, which contrasts with the tight glucose control incontinuously normal range for the patients who are critically ill.In this study lactate and pyruvate level in the coronary effluent were measuredfrom the isolated heart directly perfused with GIK and lactate. It was shown that thepreischemic lactate level was low and then clearly elevated as soon as the reperfusiontook place due to anaerobic metabolism. In accordance with reperfusion time lactate leveldecreased gradually. In relation with pyruvate level, this substrate evolution looked likethe appearance of lactate but its value was lower if compared with lactate.The recovery in functional mechanical activity of the post ischemic heart seems tobe much more related to the pattern of the evolution of logarithmic lactate/pyruvate ratio(L/P ratio). Logarithmic value of L/P ratio in GIK group increased since the earlyreperfusion period (+40%, p < 0.05), followed by improvement in recovery ofmechanical activity in this group which was significantly higher if compared with thecontrol group. Similar fashion was found in lactate group in regard to the evolution of thelogarithmic value of L/P ratio in this group, where its value was significantly highercompared with the control group. The logarithmic evolution pattern on L/P ratio for thisgroup increased along the reperfusion time (+34% p < 0.05).From the present study, it can be concluded that the recovery of functionalmechanical activity of the post ischemic heart perfused with GIK is through modificationon cellular lactate metabolism

ACE-inhibition prevents postischemic coronary leukocyte adhesion and leukocyte-dependent reperfusion injury

Objective: Polymorphonuclear leukocytes (PMN), retained in the microvascular bed, can contribute to postischemic myocardial reperfusion injury. Since a beneficial effect of ACE-inhibition on reperfusion injury has been reported, we investigated the impact of cilazaprilat on PMN dependent reperfusion injury in isolated guinea pig hearts. Methods: Hearts (n=5 per group) were subjected to 15 min of ischemia. Immediately thereafter, a bolus of PMN was injected into the coronary system. External heart work (EHW) and total cardiac nitric oxide release were measured. For microscopic evaluation, hearts received rhodamine 6G labelled PMN after ischemia, were arrested 5 min later and further perfused with FITC dextran (0.1%). Localization of retained PMN was assessed by fluorescence microscopy. Leukocyte activation was studied by FACS analysis of the adhesion molecule CD11b before and after coronary passage of the PMN. The ACE-inhibitor cilazaprilat (Cila, 2 μM) and the NO-synthase inhibitor nitro-L-arginine (NOLAG, 10 μM) were used to modulate nitric oxide formation of the heart. Results: Postischemic EHW recovered to 67±5% (controls) and 64±6% (Cila) of the preischemic value. Addition of PMN severely depressed recovery of EHW (39±2%) and NO release (39±6% of the preischemic value). Simultaneously, ischemia led to a substantial increase in postcapillary PMN adhesion (from 21±5 to 172±27 PMN/mm² surface) and CD11b-expression of the recovered PMN (3-fold). Cila attenuated postischemic PMN adhesion (83±52 PMN/mm²) and activation of PMN, whereas it improved recovery of work performance (64±4%) and NO release (65±4%) in the presence of PMN. Conversely, NOLAG increased PMN adhesion (284±40 PMN/mm²) and myocardial injury. We conclude that ACE-inhibition prevents leukocyte dependent reperfusion injury mainly by inhibition of postcapillary leukocyte adhesion. The effect may be mediated by NO, given the proadhesive effect of NOLAG