Macrophage migration inhibitory factor decreased T-type Ca2+ channel current through activating Src

Aims T-type Ca²⁺ current(I_CaT)plays an important role in the pathogenesis of atrial fibrillation（AF）.The present study sought to investigate the role of Macrophage migration inhibitory factor（MIF）,a pleiotropic cytokine,in the regulation of T-type Ca²⁺ channel in atrium myocytes.Methods We used whole-cell voltage-clamp technique and biochemical assays to study the regulation and expression of I_Ca,T in mouse atrium myocytes（HL-1 cells）.Results Serum MIF concentrations was slightly increased in patients with AF compared to sinus rhythm（SR） controls.In cultured HL-1 cells, significant amounts of MIF were produced in response to hydrogen peroxide（H₂O₂）,but not AngiotensinⅡ（AngⅡ）. Mouse recombinant MIF（rMIF）（20 or 40 nM,24 h） suppressed peak ICa,T by-38%and-60%in a concentration-dependent manner,impaired the voltage-dependent activation of I_Ca,T,and down-regulated of TCC alG mRNA.Src inhibitors genistein and PPl significantly enhanced ICaT.The depression of ICa,T induced by rMIF could be reversed by genistein and PP1.Conclusions MIFis involved in the pathogenesis of AF,probably by decreasing ICa,T through impairment of the channel function and activation of c-Src kinases in atrium myocytes.     

Changes of intra-mitochondrial Ca in adult ventricular cardiomyocytes examined using a novel fluorescent Ca indicator targeted to mitochondria

In this study a Ca²⁺ sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca²⁺-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca²⁺ sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca²⁺ transient. Cytoplasmic (Fura-2) and mitochondrial Ca²⁺ (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca²⁺ loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca²⁺ sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K_d of  220 nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarizations. Time to peak of the mitochondrial Ca²⁺ transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca²⁺ release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by  65% and increased the time to 50% decay, whilst cytosolic Ca²⁺ transients were unchanged. The mitochondrial Ca²⁺ uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca²⁺. The mitochondrial-targeted Ca²⁺ probe indicates sustained and transient phases of mitochondrial Ca²⁺ signal, which are dependent on cytoplasmic Ca²⁺ levels and require a functional mCU.     

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes

Cardiac mitochondria can take up Ca²⁺, competing with Ca²⁺ transporters like the sarcoplasmic reticulum (SR) Ca²⁺-ATPase. Rapid mitochondrial [Ca²⁺] transients have been reported to be synchronized with normal cytosolic [Ca²⁺]_i transients. However, most intra-mitochondrial free [Ca²⁺] ([Ca²⁺]_mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca²⁺]_mito in single rat myocytes using the ratiometric Ca²⁺ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca²⁺]_mito dependence on [Ca²⁺]_i (with 5 mM EGTA) was sigmoid with [Ca²⁺]_mito < [Ca²⁺]_i for [Ca²⁺]_i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca²⁺]_i (± 15 mM Na⁺) cyclical spontaneous SR Ca²⁺ release occurred (5–15/min). Changes in [Ca²⁺]_mito during individual [Ca²⁺]_i transients were small ( 2–10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca²⁺ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca²⁺]_mito and spontaneous Ca²⁺ transients (confirming that SR Ca²⁺ releases caused the [Ca²⁺]_mito rise). Confocal imaging of local [Ca²⁺]_mito (using rhod-2) showed that [Ca²⁺]_mito rose rapidly with a delay after SR Ca²⁺ release (with amplitude up to 10 nM), but declined much more slowly than [Ca²⁺]_i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca²⁺ uptake for larger [Ca²⁺]_mito transients was  0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca²⁺ buffering), consistent with prior indirect estimates from [Ca²⁺]_i measurements, and corresponds to  1% of the SR Ca²⁺ uptake during a normal Ca²⁺ transient. Thus small phasic [Ca²⁺]_mito transients and gradually integrating [Ca²⁺]_mito signals occur during repeating [Ca²⁺]_i transients.     

Influence of Infrasound Exposure on the Whole L-type Calcium Currents in Rat Ventricular Myocytes

This study was designed to examine the effect of infrasound exposure (5 Hz at 130 dB) on whole-cell L-type Ca²⁺ currents (WLCC) in rat ventricular myocytes and the underlying mechanism(s) involved. Thirty-two adult Sprague-Dawley rats were randomly assigned to infrasound exposure and control groups. [Ca²⁺]_i, WLCC, mRNA expression of the a_1c subunit of L-type Ca²⁺ channels (LCC), and SERCA2 protein were examined on day 1, 7, and 14 after initiation of infrasound exposure. Fluo-3/AM fluorescence and the laser scanning confocal microscope techniques were used to measure [Ca²⁺]_i in freshly isolated ventricular myocytes. The Ca²⁺ fluorescence intensity (FI), denoting [Ca²⁺]_i in cardiomyocytes, was significantly elevated in a time-dependent manner in the exposure groups. There was a significant increase in WLCC in the 1-day group and a further significant increase in the 7- and 14-day groups. LCC mRNA expression measured by RT-PCR revealed a significant rise in the 1-day group and a significant additional rise in the 7- and 14-day groups compared with control group. SERCA2 expression was significantly upregulated in the 1-day group followed by an overt decrease in the 7- and 14-day groups. Prolonged exposure of infrasound altered WLCC in rat cardiomyocytes by shifting the steady-state inactivation curves to the right (more depolarized direction) without altering the slope and biophysical properties of I _Ca,L. Taken together, our data suggest that changes in [Ca²⁺]_I levels as well as expression of LCC and SERCA2 may contribute to the infrasound exposure-elicited cardiac response. Zhaohui Pei and Zhiqiang Zhuang contributed equally to this work.     

Effects of benzyltetrahydropalmatine on two components of the delayed rectifier K+ current in Guinea pig ventricular myocytes

The effects of benzyltetrahydropalmatine (BTHP), a new class III antiarrhythmic agent, on the action potential in guinea pig papillary muscle and the rapidly activating component (I _Kr) and the slowly activating component (I _Ks) of the delayed rectifier potassium current (I _K) in isolated guinea pig ventricular myocytes were investigated. The action potentials of papillary muscles were studied using a standard microelectrode technique, while the K⁺ currents were recorded using the whole-cell patch clamp technique. The results showed that BTHP prolonged the action potential duration (APD) without altering other variables of the action potential in guinea pig papillary muscles. The 2 components of I _K were blocked by BTHP (1 100 mol·L^–1) in time-, concentration-, voltage-, and specifically frequency-dependent fashion. The IC₅₀ value for blockade ofI _Kr was 13.5 mol·L^–1, while the IC₅₀ value for blockade of I _Ks was 9.3 mol·L^–1. BTHP 30.0 mol·L^–1 reduced I _Kr and I _Kr,tail by 31 ± 4.3% and 36 ± 4.7% (n = 6, p < 0.01) and decreased I _Ks and I _Ks,tail by 40 ± 6.3% and 39 ± 4.6% (n = 7, p < 0.01) respectively. BTHP accelerated their deactivation course by reducing the time constants of deactivation of I _Kr and I _Ks. The activation kinetics of I _Kr or I _Ks were not affected by BTHP. It is concluded that BTHP prolonged the action potential duration with respect to its non-selective action on I _Kr and I _Ks in single guinea pig ventricular cell in a frequency-dependent fashion.     

Arrhythmias,elicited by catecholamines and serotonin,vanish in human chronic atrial fibrillation

Atrial fibrillation (AF) is the most common heart rhythm disorder. Transient postoperative AF can be elicited by high sympathetic nervous system activity. Catecholamines and serotonin cause arrhythmias in atrial trabeculae from patients with sinus rhythm (SR), but whether these arrhythmias occur in patients with chronic AF is unknown. We compared the incidence of arrhythmic contractions caused by norepinephrine, epinephrine, serotonin, and forskolin in atrial trabeculae from patients with SR and patients with AF. In the patients with AF, arrhythmias were markedly reduced for the agonists and abolished for forskolin, whereas maximum inotropic responses were markedly blunted only for serotonin. Serotonin and forskolin produced spontaneous diastolic Ca²⁺ releases in atrial myocytes from the patients with SR that were abolished or reduced in myocytes from the patients with AF. For matching L-type Ca²⁺-current (I_Ca,L) responses, serotonin required and produced ∼100-fold less cAMP/PKA at the Ca²⁺ channel domain compared with the catecholamines and forskolin. Norepinephrine-evoked I_Ca,L responses were decreased by inhibition of Ca²⁺/calmodulin-dependent kinase II (CaMKII) in myocytes from patients with SR, but not in those from patients with AF. Agonist-evoked phosphorylation by CaMKII at phospholamban (Thr-17), but not of ryanodine2 (Ser-2814), was reduced in trabeculae from patients with AF. The decreased CaMKII activity may contribute to the blunting of agonist-evoked arrhythmias in the atrial myocardium of patients with AF.Atrial fibrillation (AF) is the most common cardiac arrhythmia, associated with increased risk of death, congestive heart failure, and stroke (1). AF is characterized by an irregular, often rapid heart rate. Atria contract with reduced force, thereby favoring thrombus formation (2). AF occurs in several cardiac diseases, and its incidence is higher in woman than in men, particularly in those with valvular heart disease (SI Appendix, Table S1). Chronic AF causes structural and electrical remodeling, as well as enlarged atria (SI Appendix, Table S1), which in turn contributes to maintain AF.Atrial contractility is reduced in isolated atrial tissues (3) obtained from patients with chronic AF, attributed to a marked decrease in L-type Ca²⁺ current (I_Ca,L) (3, 4). The inotropic responses to the agonist for β₁-adrenergic receptors (β₁ARs) and β₂-adrenergic receptors (β₂ARs), isoproterenol (ISO), but not the density of βARs and G proteins, are decreased in AF (3); however, whether the function of coexisting β₁ARs and/or β₂ARs is perturbed is unknown. Activation of human atrial β₁ARs, β₂ARs, and serotonin [5-hydroxytryptamine (5-HT)] 5-HT₄ receptors hastens relaxation through phosphorylation of phospholamban (PLB) (5–7) by cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin-dependent kinase II (CaMKII) and produces arrhythmias (8, 9) in atrial trabeculae in patients with sinus rhythm (SR). Catecholamines and 5-HT have been proposed to initiate AF (8, 9). The relevance of these in vitro arrhythmias is corroborated by the clinical finding that high sympathetic nervous system activity during and after cardiac surgery causes premature beats and transient postoperative AF in approximately one-third of patients (10).Increased propensity to generate spontaneous impulses is assumed to initiate and/or maintain AF in humans. Arrhythmias may develop through spontaneous impulse generation within individual myocytes and/or reentry around nonexcitable tissue. The traveling electrical impulse of a premature atrial beat can encounter areas of refractoriness, return to its origin in a retrograde way, and through reentry initiate and maintain AF. Spontaneous impulse generation could be related to increased activity of PKA and/or CaMKII, with subsequent uncoordinated release of Ca²⁺ from the sarcoplasmic reticulum. Such a concept is attractive, because Ca²⁺ released from the “leaky” sarcoplasmic reticulum would activate the Na⁺-Ca²⁺ exchanger to extrude Ca²⁺ and to produce a arrhythmogenic depolarizing current, thereby explaining both the contractile dysfunction and the high recurrence rate (11–13).If arrhythmias were initiated and maintained by increased activity of PKA and/or CaMKII, then interventions known to stimulate both kinases would be expected to evoke more arrhythmias in patients with AF. However, in vitro induction of arrhythmias and activation of PKA and CaMKII by catecholamines and 5-HT were not assessed in tissues from patients with AF. Thus, we compared the effects of endogenous agonists on force and arrhythmias in intact trabeculae, as well as the I_Ca,L, Ca²⁺ transients (CaTs), and diastolic Ca²⁺ release, in atrial myocytes obtained from patients with SR and patients with AF. Functional measurements, including relaxation, were supplemented by Western blot analysis of relevant targets of PKA and CaMKII. Chronic AF caused a marked decrease in agonist-evoked arrhythmias, associated with a decrease in some CaMKII-catalyzed functions but unchanged PKA functions.     

The relationship between myocardial oxygen consumption and total work obtained by a new left ventricular model

Summary The relationship between myocardial oxygen consumption ( ) and left ventricular work was examined experimentally, assuming that the left ventricle is equivalent to an electrical current source generator consisting of parallel source resistance (Ri) and constant generator current (I₀). The internal and external work can be calculated as (Ri×I₁ ²) and (Rp×I₂ ²) in this model, where I₁ is current of source resistance, Rp is peripheral resistance and I₂ is cardiac output.The experiments were performed with a blood-perfused isolated ejecting canine heart preparation in which the hydraulic model, simulated to an aortic input impedance, was attached to the aortic root. The peripheral resistance was changed in a stepwise fashion and left ventricular pressure, cardiac output and were measured.The calculated total work showed a close linear correlation with the which suggested that the left ventricle can be regarded as a current source generator and the total work as a main mechanical factor in determining the .     

Regional and frequency-dependent changes in action potentials and transient outward K<Superscript>+</Superscript> currents in ventricular myocytes from J-2-K cardiomyopathic hamsters

Abstract. Objectives Although lengthening of action potential duration (APD) and decreased transient outward K⁺ currents (I_to) have been observed in ventricular myocytes from cardiomyopathic hamsters, epi- and endo-cardial differences in I_to and their roles in frequency-dependent changes in APD have not been claried. Methods The patch-clamp technique of whole-cell conguration was used to record membrane potentials and currents in epicardial and endocardial myocytes of the J-2 hamster germline without (control) and with cardiomyopathy (CM). Results In control, APD in endocardial myocytes was longer than that in epicardial myocytes at 0.1 Hz. APD significantly lengthened with increased frequencies of stimulation from 0.1 to 6.0 Hz in both groups with the longer APD in endocardial myocytes. In CM, APD lengthened in epicardial myocytes exceeding the endocardial APD without a frequency-dependent prolongation. Pretreatment with 4 mM 4-aminopyridine completely abolished the frequency-dependent changes and abolished APD differences between epicardial and endocardial myocytes, and between control and CM hamsters. The transient outward K⁺ current (I_to) significantly decreased in epicardial myocytes from CM hamsters compared with that of control (17.5 ± 1.5 pA/pF in control vs. 9.5 ± 2.5 pA/pF in CM at +60 mV) with altered recovery from inactivation, without changes in the endocardial I_to. Moreover, the inward rectifier K⁺ current decreased in epicardial myocytes from CM hamsters and the L-type Ca2⁺ current reduced in both regions from CM compared to control. Conclusion Results indicate that differences in APD between epi- and endocardial myocytes in CM hamsters are mainly caused by a decreased current density and altered recovery from inactivation of I_to in epicardial myocytes.     

Chronic obstructive sleep apnea causes atrial remodeling in canines: mechanisms and implications

Obstructive sleep apnea (OSA) is closely related to atrial fibrillation (AF). However, the roles and mechanisms of chronic OSA in atrial remodeling are still unclear. Canine model of chronic OSA was simulated by stopping the ventilator and closing the airway for 4 h per day and lasting for 12 weeks. AF inducibility and duration was increased while atrial effective refractory period (AERP) was shortened after chronic apnea. Meanwhile, upregulation of proteins encoding inward rectifier K⁺ current (I _K1), delayed rectifier K⁺ current (I _Kr and I _Ks), acetylcholine activated K⁺ current (I _KACh), transient outward K⁺ current (I _to) and ultra-rapid delayed rectifier potassium current (I _Kur) as well as downregulation of protein encoding L-type Ca²⁺ current (I _Ca,L) were found after chronic OSA. Besides abnormal electrical activity, chronic OSA induced apoptosis and interstitial fibrosis of atrial myocytes, which was partly mediated by caspase 9, phosphorylation of extracellular-regulated kinase 1/2, and α-smooth muscle actin. In addition, atrial sympathetic and parasympathetic hyperinnervation were found manifesting by enhanced growth-associated protein 43, tyrosine hydroxylase and elevated choline acetyltransferase. Moreover, protein expression of β₁, β₂, and M₂ receptor were markedly increased by chronic OSA. In summary, we firstly demonstrated in canine model that chronic OSA could shorten AERP and lead to altered expression of important channel proteins, moreover, induce atrial structure remodeling by increased atrial apoptosis, fibrosis, and autonomic remodeling, eventually promoting the development of a substrate of AF. Our findings suggested that reversing atrial remodeling might be a potential therapeutic strategy for OSA-induced AF.     

Constitutive CaMKII activity regulates Na channel in rat ventricular myocytes

The cardiac voltage-gated Na⁺ channel controls the upstroke of action potential and membrane excitability. The Na⁺ channel associates with Ca²⁺/CaM-dependent protein kinase (CaMKII), but the role of CaMKII on Na⁺ channel activity in the resting state is not clear. In this report, we investigated whether CaMKII constitutively regulates Na⁺ currents (I_Na), independent of Ca²⁺ influx in rat ventricular myocytes using patch clamp technique. CaMKII inhibition (by KN93 or autocamtide-related inhibitory peptide) caused a negative shift in I_Na steady-state inactivation and delayed recovery from slow inactivation, limiting channel availability. The reduction of I_Na was 29.47 ± 3.01% at a holding potential (V_h) of − 120 mV and it increased to 77.70 ± 7.92% when V_h was − 70 mV, suggesting that near the resting membrane potential, three-quarters of I_Na depends on CaMKII action. CaMKII inhibition also enhanced intermediate inactivation, as well as delayed recovery from fast inactivation, and decreased late I_Na. KN92, an inactive analog of KN93, had no effect on I_Na. Using an antibody against phosphorylated (activated) CaMKII, we found that constitutively active CaMKII co-immunoprecipitated with Na⁺ channels under resting conditions. CaMKII inhibitors reduced the level of phosphorylated CaMKII, which correlated with the degree of reduction in channel availability. These data suggest that CaMKII in an active form contributes to regulating I_Na. Finally, we observed a drastic reduction in the upstroke velocity of action potentials upon CaMKII inhibition. In conclusion, CaMKII constitutively regulates cardiac Na⁺ channel and this regulatory mechanism is important for the maintenance of Na⁺ channel characteristics under physiological conditions.     

Agonist-independent modulation of L-type Ca currents by basal Gs protein activities in single guinea pig ventricular myocytes

The modulation of L-type Ca²⁺ currents (I _Ca,L) by the basal activities of G proteins was studied in adult guinea pig ventricular myocytes by whole-cell patch-clamp techniques. With intrapipette guanosine triphosphate (GTP) (100 μM), a specific inhibition of G_i proteins by pertussis toxin (PTX) produced an increase in the basal density of I _Ca,L (from 11.0 ± 0.8, n = 13, to 25.0 ± 2.0 pA/pF, n = 11, at 0 mV test potential). In addition, PTX shifted the forskolin (Fsk) concentration–I _Ca,L response relation significantly leftward (EC₅₀ = 63.7 ± 12.5 vs 625 ± 75 nM). With intrapipette guanosine diphosphate (GDP)βS (1 mM), the Fsk–I _Ca,L relation was also shifted leftward (EC₅₀ = 197 ± 18.3 vs 781 ± 82.5 nM). However, chronic GDPβS dialysis accelerated the rundown of I _Ca,L significantly, suggesting a potential contribution of G_s proteins in maintaining basal I _Ca,L. In contrast, intra-pipette GTPγS (100 μM) produced a transient rise in I_Ca,L from 11.0 ± 3.0 to 22.8 ± 7.0 pA/pF (in 3.4 min after whole-cell formation at 0 mV, n = 9), presumably through the activation of G_s proteins. It was followed by a gradual decline in I _Ca,L (to 15.5 ± 3.5 pA/pF), which was still enhanced by Fsk (EC₅₀ = 1450 ± 98 nM), indicating that the current decay was not solely due to rundown but to activation of G_i proteins. G_s, in addition to G_i proteins, show sufficient basal activity to modulate I _Ca,L in an agonist-independent manner. Received: October 19, 2000 / Accepted: February 24, 2001     

Role of reactive oxygen species (ROS) in angiotensin II-induced stimulation of the cardiac Na/HCO3 cotransport

The sarcolemmal Na⁺/HCO₃⁻ cotransporter (NBC) plays an important role in intracellular pH (pH_i) regulation in the heart. In the present work we studied, in isolated cat ventricular myocytes, the role of Angiotensin II (Ang II) and reactive oxygen species (ROS) production as potential activators of the NBC. pH_i was measured in single cells in a medium with HCO₃⁻ using the fluorescent pH indicator BCECF. The NH₄⁺ pulse method was used to induce an intracellular acid load and the acid efflux (J_H) in the presence of the Na⁺/H⁺ exchanger blocker HOE642 (10 μM) was calculated as indicator of NBC activity. The following J_H data are presented at pH_i of 6.8 ( and ^# indicate p < 0.05 after ANOVA vs. control and Ang II, respectively). The basal J_H (1.03 ± 0.12 mM/min, n = 11) was significantly increased in the presence of 100 nM Ang II (1.70 ± 0.15 mM/min, n = 8). This effect of Ang II was abolished when we added to the extracellular solution 2 mM MPG (ROS scavenger; 0.80 ± 0.08 mM/min, n = 11^#), 300 μM apocynin (NADPH oxidase blocker; 0.80 ± 0.13 mM/min, n = 6^#), 500 μM 5-hydroxidecanoate (mitochondrial ATP dependent K⁺ channel, mK_ATP, blocker; 0.97 ± 0.21 mM/min, n = 9^#), or the inhibitor of the MAP kinase ERK pathway U0126 (10 μM; 0.56 ± 0.18 mM/min, n = 6^#). We also determined the phosphorylation of ERK during the first min of acidosis and we detected that Ang II significantly enhanced the ERK phosphorylation levels, an effect that was cancelled by scavenging ROS with MPG. In conclusion, we propose that Ang II enhances the production of ROS through the activation of the NADPH oxidase, which in turn triggers mK_ATP opening and mitochondrial ROS production (“ROS-induced ROS-release mechanism”). Finally, these mitochondrial ROS stimulate the ERK pathway, leading to the activation of the NBC.     

Exogenous Administration of Recombinant MIF at Physiological Concentrations Failed to Attenuate Infarct Size in a Langendorff Perfused Isolated Mouse Heart Model

Purpose

Evidence suggests a two-pronged role of endogenous macrophage migration inhibitory factor (MIF) release in ischemia/reperfusion injury. We aimed to assess whether its exogenous administration confers cardioprotection.

Methods

Male C57/BL6 mice were randomly allocated to receive recombinant mouse MIF (rMIF) at physiological (ng/mL) concentrations in a dose–response fashion before or after a protocol of 35 min of ischemia and 2 h of reperfusion in an isolated Langendorff-perfused model with infarct size as endpoint. Isolated primary cardiomyocytes were also used for cell survival studies using rMIF at a supra-physiological concentration of 1 μg/mL. Pro-survival kinase activation was also studied using Western blot analyses.

Results

Exogenous MIF did not elicit a cardioprotective effect either when administered before the ischemic insult or when applied at reperfusion. rMIF did not confer protection when it was applied immediately before or after a hypoxia/reoxygenation insult in primary isolated cardiomyocytes. Consistently, hearts treated with MIF did not show a significant increase in phosphorylated Akt and ERK1/2.

Conclusion

The exogenous administration of rMIF in a physiological concentration range both before ischemia and at reperfusion did not show cardioprotective effects. Although these results do not address the role of endogenous MIF after an ischemic insult followed by reperfusion, they may limit the potential translational value of rMIF.

     

The new selective IKs–blocking agent HMR 1556 restores sinus rhythm and prevents heart failure in pigs with persistent atrial fibrillation

Background Antiarrhythmic drugs for treatment of atrial fibrillation in patients with heart failure are limited by proarrhythmia and low efficacy. Experimental studies indicate that the pure I_Ks blocking agents chromanol 293b and HMR 1556 prolong repolarization more markedly at fast than at slow heart rates and during –adrenergic stimulation. These properties may overcome some of the above quoted limitations. Methods and results Ten domestic swine underwent pacemaker implantation (PM) and atrial burst pacing to induce persistent AF. Four days after onset of persistent AF, pigs were randomized to HMR 1556 (30 mg/kg, p.o., 10 days) or placebo. All animals receiving HMR 1556 converted to SR (5.2 ± 1.9 days), whereas placebo pigs remained in AF. Pigs treated with placebo developed high ventricular rates (297 ± 5 bpm) and severe heart failure, whereas pigs treated with HMR 1556 remained hemodynamically stable. Left ventricular ejection fraction on the day of euthanization was significantly lower in the placebo compared to the HMR 1556 group (30 ± 4% vs. 69 ± 5%, p < 0.005). Similar results were seen with epinephrine levels (placebo 1563 ± 193 pmol/l vs. HMR 613 ± 196 pmol/l, p < 0.05). Right atrial monophasic action potentials were significantly longer in the HMR 1556 compared to the placebo group (230 ± 7 ms vs. 174 ± 13 ms, p < 0.05). Conclusions The new I_Ks blocker HMR 1556 efficiently and safely restores SR and prevents CHF in a model of persistent AF. Restoration of SR is most likely linked to a marked prolongation of atrial repolarization even at high heart rates.     

Reduced DIDS-sensitive chloride conductance in Ae1-/- mouse erythrocytes

The resting membrane potential of the human erythrocyte is largely determined by a constitutive Cl⁻ conductance  100-fold greater than the resting cation conductance. The 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive electroneutral Cl⁻ transport mediated by the human erythroid Cl⁻/HCO₃⁻ exchanger, AE1 (SLC4A1, band 3) is > 10,000-fold greater than can be accounted for by the Cl⁻ conductance of the red cell. The molecular identities of conductive anion pathways across the red cell membrane remain poorly defined. We have examined red cell Cl⁻ conductance in the Ae1^−/− mouse as a genetic test of the hypothesis that Ae1 mediates DIDS-sensitive Cl⁻ conductance in mouse red cells. We report here that wildtype mouse red cell membrane potential resembles that of human red cells in the predominance of its Cl⁻ conductance. We show with four technical approaches that the DIDS-sensitive component of erythroid Cl⁻ conductance is reduced or absent from Ae1^−/− red cells. These results are consistent with the hypothesis that the Ae1 anion exchanger polypeptide can operate infrequently in a conductive mode. However, the fragile red cell membrane of the Ae1^−/− mouse red cell exhibits reduced abundance or loss of multiple polypeptides. Thus, loss of one or more distinct, DIDS-sensitive anion channel polypeptide(s) from the Ae1^−/− red cell membrane cannot be ruled out as an explanation for the reduced DIDS-sensitive anion conductance.     

Oxidoreductase regulation of Kv currents in rat ventricle

Oxidative stress contributes to the arrhythmogenic substrate created by myocardial ischemia–reperfusion partly through a shift in cell redox state, a key modulator of protein function. The activity of many oxidation-sensitive proteins is controlled by oxidoreductase systems that regulate the redox state of cysteine thiol groups, but the impact of these systems on ion channel function is not well defined. Thus, we examined the roles of the thioredoxin and glutaredoxin systems in controlling K⁺ channels in the ventricle. An oxidative shift in redox state was elicited in isolated rat ventricular myocytes by brief exposure to diamide, a thiol-specific, membrane-permeable oxidant. Voltage-clamp studies showed that diamide decreased peak outward K⁺ current (I_peak) evoked by depolarizing test pulses by 41% (+ 60 mV; p < 0.05) while steady-state outward current (I_ss) measured at the end of the test pulse was decreased by 45% (p < 0.05). These electrophysiological effects were not prevented by protein kinase C blockers, but the tyrosine kinase inhibitors genistein or lavendustin A blocked the suppression of both K⁺ currents by diamide. Moreover, inhibition of I_peak and I_ss by diamide was reversed by dichloroacetate and an insulin-mimetic. The effect of dichloroacetate to normalize I_peak after diamide was blocked by the thioredoxin system inhibitors auranofin or 13-cis-retinoic acid, but I_ss was not affected by either compound. A pan-specific inhibitor of glutaredoxin and thioredoxin systems, 1,3-bis-(2-chloroethyl)-1-nitrosourea, also blocked the dichloroacetate effect on I_peak but only partially inhibited the recovery of I_ss. These data suggest that acute regulation of cardiac K⁺ channels by oxidoreductase systems is mediated by redox-sensitive tyrosine kinase/phosphatase pathways. The pathways controlling I_peak channels are targets of the thioredoxin system whereas those regulating I_ss channels are likely controlled by the glutaredoxin system. Thus, cardiac oxidoreductase systems may be important regulators of ion channels affected by pathogenic oxidative stress.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Prognostic value of spontaneous hyponatremia in cirrhosis with ascites

Spontaneous hyponatremia in cirrhosis with ascites is generally considered to be due to an impaired renal ability to excrete free water, to be a contraindication of diuretics, and to be a bad prognostic sign. These concepts are reviewed in this paper. 55 cirrhotics with ascites were divided into three groups. Group I consisted of 13 patients with hyponatremia and very low free-water clearance ( , 0.07±0.26 ml/min). These patients also had poor renal function: low inulin clearance (C _INU , 40.6±25.9 ml/min) and paraamino-hippurate clearance (C _PAH , 383±275 ml/min). Group II consisted of 8 patients who also had hyponatremia. , C _INU , and C _PAH in these patients were fairly high: 5.85±1.53 ml/min, 85.7±26.2 ml/min, and 651±294 ml/min. These values are similar to those of the 34 patients without hyponatremia who make up Group III: (6.37±4.27 ml/min, 94.7±33.1 ml/min, and 598±199 ml/min). Hyponatremia in Group I could be related to the impaired free-water clearance. The mechanism of hyponatremia in Group II patients is not clear. Patients with hyponatremia and low C _INU and C _PAH had a negative response to diuretics and a poor prognosis. Patients with hyponatremia but with relatively good renal function had a good prognosis, similar to Group III patients. They responded to diuretics with no worsening of their hyponatremia.Presented in part at the Eighth Meeting of the European Association for the Study of the Liver, September, 1973, Vittel, France.     

Effects of caffeine on energy output of rabbit heart muscle

Summary The effects of caffeine (1 mmol·1^–1) on mechanical and energetic parameters in the arterially perfused interventricular rabbit septa were examined at various frequencies of stimulation. Even though 1 mmol^–1 caffeine induced a negative inotropic effect only at stimulation rates higher than 0.33 Hz. relaxation was impaired at all frequencies tested. The ratio between maximum rate of relaxation and developed tension was consistently lowered by caffeine, indicating a more marked effect on relaxation over contraction. In addition, while time-to-peak tension was unaffected by caffeine at the dose used, the last part of the relaxation (i.e., of the contractile event) was prolonged at all frequencies in the presence of the drug. Resting heat production (H _t ) was increased in the presence of caffeine (1.6±0.6 mW·g^–1). The ratios between active heat production and either developed tension (H_a/T) or tension time integral (H_a/TtI), increased at all frequencies examined (53.3±8.5 J·mN^–1·g^–1 and 68.2±9.9 J·mN^–1·s^–1·g^–1, respectively), indicating a lowered economy of the contractile process. This is consistent with the lower ATP/Ca ratio reported for the sarcoreticular Ca pump (i.e., one ATP hydrolyzed/2 Ca transported) with respect to the sarcolemmal mechanisms such as Na–Ca exchanger or the sarcolemmal Ca pump, with an ATP/Ca ratio of 1 to 1. Thus, inhibition of the SR-Ca pump by caffeine would induce a higher rate of ATP hydrolysis with the consequent increase in the H_a/T ratio. As a result of the increase in both H_a/T ratio and H_r induced by caffeine, the ratio between total heat production and developed tension (H_t/T) also increased. Therefore, the contractile process appeared to be more efficient in the presence of an active SR, since it is energetically less costly to generate a given level of isometric tension.     

Oxidant-stimulated chloride secretion in rat jejunum in vitro is mediated by eicosanoids

Specific effects of the cytotoxic secondary lipid oxidation product, 4-hydroxynonenal (10^–8–10^–4 M), on intact sheets of rat jejunum were measured as changes in short circuit current (I_sc) following cumulative addition to either the mucosal or serosal side, using the analogous aldehyde, nonenal, as reference. 4-Hydroxynonenal stimulated I_sc from the serosal side (maximal I_sc = 27.2 ± 3.5 A/cm², mean ± SEM, N = 32) while nonenal stimulated I_sc primarily from the mucosal side (maximal I_sc = 16.2 ± 3.4 A/cm², N = 20). Inhibition by 100 M bumetanide (4-hydroxynonenal: 88.9 ± 3.0%, N = 6, p < 0.05, nonenal: 69.3 ± 2.9%, N = 6, P < 0.05) indicated chloride secretion. Nonenal-induced I_sc was inhibited (72.5 ± 1.2%, N = 8, P < 0.05) by a combination of nordihydroguaiaretic acid (100 M) and piroxicam (10 M), while 4-hydroxynonenal-induced I_sc, was abolished by piroxicam (N = 8, P < 0.001) and inhibited by 1 M tetrodotoxin (69.8 ± 9.7%, N = 6, P < 0.001). These data indicate that 4-hydroxynonenal stimulates chloride secretion mediated by prostaglandins and the enteric nervous system. The site of action (serosal) being opposite to the reference aldehyde.