Krause Eg

Identification and Expression of δ-Isoforms of the Multifunctional Ca2+/Calmodulin-Dependent Protein Kinase in Failing and Nonfailing Human Myocardium

Despite its importance for the regulation of heart function, little is known about the isoform expression of the multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII) in human myocardium. In this study, we investigated the spectrum of CaMKII isoforms delta2, delta3, delta4, delta8, and delta9 in human striated muscle tissue. Isoform delta3 is characteristically expressed in cardiac muscle. In skeletal muscle, specific expression of a new isoform termed delta11 is demonstrated. Complete sequencing of human delta2 cDNA, representing all common features of the investigated CaMKII subclass, revealed its high homology to the corresponding rat cDNA. Comparative semiquantitative reverse transcription-polymerase chain reaction analyses from left ventricular tissues of normal hearts and from patients suffering from dilated cardiomyopathy showed a significant increase in transcript levels of isoform delta3 relative to the expression of glyceraldehyde-3-phosphate dehydrogenase in diseased hearts (101. 6+/-11.0% versus 64.9+/-9.9% in the nonfailing group; P<0.05, n=6). Transcript levels of the other investigated cardiac CaMKII isoforms remained unchanged. At the protein level, by using a subclass-specific antibody, we observed a similar increase of a delta-CaMKII-specific signal (7.2+/-1.0 versus 3.8+/-0.7 optical density units in the nonfailing group; P<0.05, n=4 through 6). The diseased state of the failing hearts was confirmed by a significant increase in transcript levels for atrial natriuretic peptide (292. 9+/-76.4% versus 40.1+/-3.2% in the nonfailing group; P<0.05, n=3 through 6). Our data characterize for the first time the delta-CaMKII isoform expression pattern in human hearts and demonstrate changes in this expression pattern in heart failure.

Regulation of the Transient Outward K+ Current by Ca2+/Calmodulin-Dependent Protein Kinases II in Human Atrial Myocytes

Ca(2+)/calmodulin-dependent protein kinases II (CaMKII) have important functions in regulating cardiac excitability and contractility. In the present study, we examined whether CaMKII regulated the transient outward K(+) current (I(to)) in whole-cell patch-clamped human atrial myocytes. We found that a specific CaMKII inhibitor, KN-93 (20 micromol/L), but not its inactive analog, KN-92, accelerated the inactivation of I(to) (tau(fast): 66.9+/-4.4 versus 43.0+/-4.4 ms, n=35; P<0.0001) and inhibited its maintained component (at +60 mV, 4.9+/-0.4 versus 2.8+/-0.4 pA/pF, n = 35; P<0. 0001), leading to an increase in the extent of its inactivation. Similar effects were observed by dialyzing cells with a peptide corresponding to CaMKII residues 281 to 309 or with autocamtide-2-related inhibitory peptide and by external application of the calmodulin inhibitor calmidazolium, which also suppressed the effects of KN-93. Furthermore, the phosphatase inhibitor okadaic acid (500 nmol/L) slowed I(to) inactivation, increased I(sus), and inhibited the effects of KN-93. Changes in [Ca(2+)](i) by dialyzing cells with approximately 30 nmol/L Ca(2+) or by using the fast Ca(2+) buffer BAPTA had opposite effects on I(to). In BAPTA-loaded myocytes, I(to) was less sensitive to KN-93. In myocytes from patients in chronic atrial fibrillation, characterized by a prominent I(sus), KN-93 still increased the extent of inactivation of I(to). Western blot analysis of atrial samples showed that delta-CaMKII expression was enhanced during chronic atrial fibrillation. In conclusion, CaMKII control the extent of inactivation of I(to) in human atrial myocytes, a process that could contribute to I(to) alterations observed during chronic atrial fibrillation.

Metabolic control characteristics of the acutely ischemic myocardium

HE BEATING warm-blooded heart cannot tolerate ischemia and the ensuing hypoxia of its tissue for more than a short time-one minute at most” without serious impairment of its function. From the point of view of the energy needs of the musc:le, this is the case not so much because the myocardial energy reserves, consisting chiefly of glycogen, are limited and may become exhausted. It is rather because the rate at which glycogen can be metabolized, via glucosel-phosphate and the glycolytic pathway, is not high enough in heart muscle to make up, in the face of continued utilization of phosphate bond energy, for the low efficiency of glycolysis in synthesizing adenosine triphosphate (ATP).2 Energetically speaking, the ischemic warmblooded heart or heart muscle takes a downhill course without reaching a steady state. Nevertheless, the energy furnished by glycolysis can help the heart to overcome brief spells of myocardial ischemia and hypoxia. The regulation of myocardial glycolytic activity is therefore a problem that is not merely of theoretical interest. In the study of this problem much has been learned by examining the changes occurring in cardiac muscle during the transition from the aerobic to the anaerobic state. The present paper is a review of studies and an account of experiments utilizing this approach. For the purpose of the following discussion it is appropriate to recall3 that the essential factors determining the output of an enzyme or enzyme system at a given temperature are (1) the amount of enzyme protein; (2) the kinetic parameters of the enzyme or enzymes; and (3) the concentration of substrates and products of the reactions. Alterations of these factors constitute the means whereby control of cellular metabolism is achieved. Effecters eliciting these alterations may be constituents of the cell in question, in which case one can speak of internal control, or they may be hormones or other substances of exogenous origin. Both classes of agents have been found to operate in the heart during the transition from aerobiosis to ischemia and hypoxia. The analysis of their interaction and of the associated metabolic changes has been the subject of previous review4-6 and is carried on here to the point where one may discern in the acutely ischemic myocardium a distinct pattern of metabolic control.

β2-Adrenergic cAMP Signaling Is Uncoupled From Phosphorylation of Cytoplasmic Proteins in Canine Heart

BACKGROUND Recent studies of beta-adrenergic receptor (beta-AR) subtype signaling in in vitro preparations have raised doubts as to whether the cAMP/protein kinase A (PKA) signaling is activated in the same manner in response to beta2-AR versus beta1-AR stimulation. METHODS AND RESULTS The present study compared, in the intact dog, the magnitude and characteristics of chronotropic, inotropic, and lusitropic effects of cAMP accumulation, PKA activation, and PKA-dependent phosphorylation of key effector proteins in response to beta-AR subtype stimulation. In addition, many of these parameters and L-type Ca2+ current (ICa) were also measured in single canine ventricular myocytes. The results indicate that although the cAMP/PKA-dependent phosphorylation cascade activated by beta1-AR stimulation could explain the resultant modulation of cardiac function, substantial beta2-AR-mediated chronotropic, inotropic, and lusitropic responses occurred in the absence of PKA activation and phosphorylation of nonsarcolemmal proteins, including phospholamban, troponin I, C protein, and glycogen phosphorylase kinase. However, in single canine myocytes, we found that beta2-AR-stimulated increases in both ICa and contraction were abolished by PKA inhibition. Thus, the beta2-AR-directed cAMP/PKA signaling modulates sarcolemmal L-type Ca2+ channels but does not regulate PKA-dependent phosphorylation of cytoplasmic proteins. CONCLUSIONS These results indicate that the dissociation of beta2-AR signaling from cAMP regulatory systems is only apparent and that beta2-AR-stimulated cAMP/PKA signaling is uncoupled from phosphorylation of nonsarcolemmal regulatory proteins involved in excitation-contraction coupling.

Activation of β2-Adrenergic Receptors Hastens Relaxation and Mediates Phosphorylation of Phospholamban, Troponin I, and C-Protein in Ventricular Myocardium From Patients With Terminal Heart Failure

BACKGROUND Catecholamines hasten cardiac relaxation through beta-adrenergic receptors, presumably by phosphorylation of several proteins, but it is unknown which receptor subtypes are involved in human ventricle. We assessed the role of beta1- and beta2-adrenergic receptors in phosphorylating proteins implicated in ventricular relaxation. METHODS AND RESULTS Right ventricular trabeculae, obtained from freshly explanted hearts of patients with dilated cardiomyopathy (n=5) or ischemic cardiomyopathy (n=5), were paced at 60 bpm. After measurement of the contractile and relaxant effects of epinephrine (10 micromol/L) or zinterol (10 micromol/L), mediated through beta2-adrenergic receptors, and of norepinephrine (10 micromol/L), mediated through beta1-adrenergic receptors, tissues were freeze clamped. We assessed phosphorylation of phospholamban, troponin I, and C-protein, as well as specific phosphorylation of phospholamban at serine 16 and threonine 17. Data did not differ between the 2 disease groups and were therefore pooled. Epinephrine, zinterol, and norepinephrine increased contractile force to approximately the same extent, hastened the onset of relaxation by 15+/-3%, 5+/-2%, and 20+/-3%, respectively, and reduced the time to half-relaxation by 26+/-3%, 21+/-3%, and 37+/-3%. These effects of epinephrine, zinterol, and norepinephrine were associated with phosphorylation (pmol phosphate/mg protein) of phospholamban 14+/-3, 12+/-4, and 12+/-3; troponin I 40+/-7, 33+/-7, and 31+/-6; and C-protein 7.2+/-1.9, 9.3+/-1.4, and 7.5+/-2.0. Phosphorylation of phospholamban occurred at both Ser16 and Thr17 residues through both beta1- and beta2-adrenergic receptors. CONCLUSIONS Norepinephrine and epinephrine hasten human ventricular relaxation and promote phosphorylation of implicated proteins through both beta1- and beta2-adrenergic receptors, thereby potentially improving diastolic function.

Phosphorylation of the L‐type calcium channel β subunit is involved in β‐adrenergic signal transduction in canine myocardium

Cyclic AMP‐mediated phosphorylation of calcium channel submits was studied in vitro and in vivo in preparations from dog heart. Calcium channels in native cardiac membranes were phosphorylated by cAMP‐dependent protein kinase (PKA) solubilized with digitonin and subsequently immunoprecipitated using a polyclonal antibody generated against the deduced carboxy‐terminal sequence of the cardiac β subunit. A 62 kDa protein was identified as the major PKA‐substrate in the immunoprecipitates. In the intact myocardium, this putative β subunit was found to be phosphorylated in response to cAMP elevating agents. In contrast, no phosphorylation of a protein with an electrophoretic mobility similar to the α1 subunit was detected, although 1,4‐dihydropyridine receptor sites were recovered in the immunoprecipitates. Thus, we suggest that PKA‐mediated phosphorylation of the β subunit is the major mechanism for β‐adrenergic regulation of cardiac L‐type calcium channel activity.

Cyclic changes in levels of cyclic AMP and cyclic GMP in frog myocardium during the cardiac cycle.

Cyclic AMP and cyclic GMP were measured in the ventricles of frog hearts that had been instantly frozen in situ by an automatic device at six predetermined points of the electrocardiogram. Cyclic AMP levels rose and cyclic GMP levels fell during the first three quarters of the R - T interval, which is a period corresponding to the latency phase of contraction and early phase of mechanical systole. At the time of the crest of the T wave, just after the ventricles had begun to relax, both nucleotides had returned to the preceding diastolic levels.

Ser16 prevails over Thr17 phospholamban phosphorylation in the β-adrenergic regulation of cardiac relaxation

Phospholamban is a critical regulator of sarcoplasmic reticulum Ca2+-ATPase and myocardial contractility. To determine the extent of cross signaling between Ca2+ and cAMP pathways, we have investigated the β-adrenergic-induced phosphorylation of Ser16 and Thr17 of phospholamban in perfused rat hearts using antibodies recognizing phospholamban phosphorylated at either position. Isoproterenol caused the dose-dependent phosphorylation of Ser16 and Thr17 with strikingly different half-maximal values (EC50 = 4.5 ± 1.6 and 28.2 ± 1.4 nmol/l, respectively). The phosphorylation of Ser16 induced by isoproterenol, forskolin, or 3-isobutyl-1-methylxanthine correlated to increased cardiac relaxation ( r = 0.96), whereas phosphorylation of Thr17 did not. Elevation of extracellular Ca2+did not induce phosphorylation at Thr17; only in the presence of a submaximal dose of isoproterenol, phosphorylation at Thr17 increased eightfold without additional effects on relaxation rate. Thr17 phosphorylation was partially affected by ryanodine and was completely abolished in the presence of 1 μmol/l verapamil or nifedipine. The data indicate that 1) phosphorylation of phospholamban at Ser16 by cAMP-dependent protein kinase is the main regulator of β-adrenergic-induced cardiac relaxation definitely preceding Thr17 phosphorylation and 2) the β-adrenergic-mediated phosphorylation of Thr17 by Ca2+-calmodulin-dependent protein kinase required influx of Ca2+through the L-type Ca2+ channel.

Dominant rats are natural risk takers and display increased motivation for food reward.

Risk-taking behavior is a vital aspect mediating the formation of social structure in animals. Here, we utilized the visible burrow system (VBS), a model in which rats form dominance hierarchies, to test the hypothesis that dominant rats in the VBS are natural risk takers and display an increased motivational state after VBS exposure. In particular, we predicted that dominant rats would have attenuated anxiety-like behavior and augmented acquisition of operant responding for food reward relative to subordinate and controls. We further hypothesized that these behaviors would correlate with elevated mesocortical orexin signaling. Prior to burrow exposure, male Long-Evans rats were tested on the elevated plus maze (EPM), and subsequently exposed to the VBS for seven consecutive days. At the conclusion of burrow exposure body weight and plasma corticosterone were used to confirm social rank within each colony. Interestingly, rats that went on to become dominant in the VBS spent significantly more time in the open arms of the EPM prior to burrow exposure and displayed increased operant responding for food reward. This effect was present over a range of reinforcement schedules and also persisted for up to 1 month following VBS exposure. Moreover, dominant rats displayed increased orexin receptor mRNA in the medial prefrontal cortex (mPFC) relative to subordinate and control rats. These data support previous findings from our group and are consistent with the hypothesis that risk-taking behavior may precede dominance formation in social hierarchies.

In-vivo phosphorylation of the cardiac L-type calcium channel beta-subunit in response to catecholamines

In canine myocardium, the β-subunit of the L-type Ca2+ channel is phosphorylated by cAMP dependent protein kinase in vitro as well as in vivo (Haase et al. FEBS Lett 335: 217–222, 1993). We have assessed the identity of the β-subunit as well as its in vivo phosphorylation in representative experimental groups of catecholamine-challenged canine hearts. Adrenergic stimulation by high doses of both noradrenaline and isoprenaline induced rapid (within 20 sec) and nearly complete phosphorylation of the Ca2+ channel β-subunit. Phosphorylation in vivo was about 4-fold higher as compared to untreated controls. When related to catecholamine-depleted (reserpine-treated) hearts noradrenaline and isoprenaline increased the in vivo phosphorylation of the β-subunit even 8-fold. This phosphorylation correlated positively with tissue levels of cAMP, endogenous particulated cAMP-dependent protein kinase (PKA) and the rate of contractile force development dP/dtmax. The results imply the involvement of a PKA-mediated phosphorylation of the Ca2+ channel β-subunit in the adrenergic stimulation of intact canine myocardium.