Phosphorylation of phospholamban at threonine-17 reduces cardiac adrenergic contractile responsiveness in chronic pressure overload-induced hypertrophy

Physiological hemodynamic stress, such as aerobic exercise, is intermittent and requires an increase in Ca2+ -dependent contractility through sympathetic nervous system activation. Pathological hemodynamic stress, such as hypertension, is persistent and requires sustained increases in cardiac function. Over time, this causes left ventricular hypertrophy (LVH)-reduced responsiveness to sympathetic stimulation. In this study, we examined the hypothesis that blunted in vivo adrenergic contractile responsiveness in pressure overload (PO)-induced cardiac hypertrophy is caused by abnormalities in the abundance and/or basal phosphorylation state of Ca2+ regulatory proteins. PO, induced by aortic constriction, caused concentric LVH or dilated LVH. Only animals with dilation exhibited a decrease in baseline left ventricle function [fractional area change (FAC); measured with echocardiography]. All PO animals had a reduced contractile response to adrenergic agonists (increase in FAC with 40 microg.kg(-1).min(-1) dobutamine, control 0.30 +/- 0.04, n = 5 vs. banded 0.10 +/- 0.03, n = 10; P < 0.01). PO animals had reduced phospholamban (PLB) protein abundance (P = 0.07, not significant) and increased PLB phosphorylation at the calmodulin-dependent kinase II (CaMKII)-specific site (PLB-Thr17, P < 0.05) but not at the protein kinase A-specific site (PLB-Ser16). PLB-Thr17 phosphorylation was inversely correlated with dobutamine-induced increases in contractility in PO animals (r2 = 0.81, P < 0.05). Continuous induction of Ca2+ transients in isolated ventricular myocytes for 24 h increased phosphorylation at PLB-Thr17 and diminished inotropic responsiveness and PLB-Ser16 phosphorylation after exposure to isoproterenol (P < 0.05). These data show that reduced adrenergic responsiveness in feline PO hypertrophy and failure involves increases in basal PLB-Thr17 phosphorylation, suggesting that activation of CaMKII in PO hypertrophy contributes to defective adrenergic reserve in compensated LVH and early heart failure.     

Reduced inhibitor 1 and 2 activity is associated with increased protein phosphatase type 1 activity in left ventricular myocardium of one-kidney, one-clip hypertensive rats

In failing hearts, although protein phosphatase type 1 (PP1) activity has increased, information about the regulation and status of PP1 inhibitor-1 (INH-1) and inhibitor-2 (INH-2) is limited. In this study, we examined activity and protein expression of PP1, INH-1 and INH-2 and phosphorylation of sarcoplasmic reticulum (SR) phospholamban (PLB), a substrate of PP1 and modulator of SR Ca2+-ATPase activity, in failing and non-failing hearts. These studies were performed in LV myocardium of seven rats with chronic renal hypertension produced by Goldblatt's one-kidney, one-clip procedure and seven age-matched sham-operated normal controls (CTR). Eight weeks after surgery, LV ejection fraction, LV hypertrophy, and pulmonary congestion were determined in all rats. PP1 activity (nmol 32P/min/mg non-collagen protein) was assessed in LV homogenates using 32P-labeled phosphorylase a as substrate. INH-1 and INH-2 activity was determined in the immunoprecipitate of LV homogenates and expressed as percentage inhibitory activity. Using a specific antibody, LV tissue levels of PP1C and calsequestrin (CSQ), a SR calcium binding protein, which is not altered in failing hearts, were also determined. Further, total and phosphorylated PLB, INH-1 and INH-2 protein levels were determined in the LV homogenate and phosphoprotein-enriched fraction, respectively. The band density of each protein was quantified in densitometric units and normalized to CSQ. Results: rats with chronic renal hypertension exhibited significantly reduced LV ejection fraction and increased LV hypertrophy and pulmonary congestion, characteristics of chronic heart failure (CHF). We found that compared to CTR, (1) both INH-1 (10.2+/-2 versus 57.5+/-1; p < 0.05) and INH-2 activity (3.8+/-0.4 versus 36.2+/-4; p < 0.05) were reduced, (2) total and phosphorylated PLB amount reduced, (3) protein level of phosphorylated INH-1 was reduced (2.32+/-0.1 versus 0.73+/-0.04; p < 0.05) whereas that of phosphorylated INH-2 increased (3.05+/-0.3 versus 1.42+/-0.1; p < 0.05), and (4) PP1 activity was increased approximately 2.6-fold in rats with CHF (1.59+/-0.05 versus 0.61+/-0.01; p < 0.05) while protein level of the catalytic subunit of PP1 (PP1C) increased 3.85-fold (0.77+/-0.05 versus 0.20+/-0.02; p < 0.05). These results suggest that reduced inhibitory INH-1 and INH-2 activity, increased PP1C protein level, and reduced PLB phosphorylation are associated with increased PP1 activity in failing hearts.     

Reduced inhibitor 1 and 2 activity is associated with increased protein phosphatase type 1 activity in left ventricular myocardium of one-kidney,one-clip hypertensive rats

In failing hearts, although protein phosphatase type 1 (PP1) activity has increased, information about the regulation and status of PP1 inhibitor-1 (INH-1) and inhibitor-2 (INH-2) is limited. In this study, we examined activity and protein expression of PP1, INH-1 and INH-2 and phosphorylation of sarcoplasmic reticulum (SR) phospholamban (PLB), a substrate of PP1 and modulator of SR Ca²⁺-ATPase activity, in failing and non-failing hearts. These studies were performed in LV myocardium of seven rats with chronic renal hypertension produced by Goldblatts one-kidney, one-clip procedure and seven age-matched sham-operated normal controls (CTR). Eight weeks after surgery, LV ejection fraction, LV hypertrophy, and pulmonary congestion were determined in all rats. PP1 activity (nmol ³²P/min/mg non-collagen protein) was assessed in LV homogenates using ³²P-labeled phosphorylase a as substrate. INH-1 and INH-2 activity was determined in the immunoprecipitate of LV homogenates and expressed as percentage inhibitory activity. Using a specific antibody, LV tissue levels of PP1C and calsequestrin (CSQ), a SR calcium binding protein, which is not altered in failing hearts, were also determined. Further, total and phosphorylated PLB, INH-1 and INH-2 protein levels were determined in the LV homogenate and phosphoprotein-enriched fraction, respectively. The band density of each protein was quantified in densitometric units and normalized to CSQ. Results: rats with chronic renal hypertension exhibited significantly reduced LV ejection fraction and increased LV hypertrophy and pulmonary congestion, characteristics of chronic heart failure (CHF). We found that compared to CTR, (1) both INH-1 (10.2 ± 2 versus 57.5 ± 1; p<0.05) and INH-2 activity (3.8 ± 0.4 versus 36.2 ± 4; p<0.05) were reduced, (2) total and phosphorylated PLB amount reduced, (3) protein level of phosphorylated INH-1 was reduced (2.32 ± 0.1 versus 0.73 ± 0.04; p<0.05) whereas that of phosphorylated INH-2 increased (3.05 ± 0.3 versus 1.42 ± 0.1; p<0.05), and (4) PP1 activity was increased approximately 2.6-fold in rats with CHF (1.59 ± 0.05 versus 0.61 ± 0.01; p<0.05) while protein level of the catalytic subunit of PP1 (PP1C) increased 3.85-fold (0.77 ± 0.05 versus 0.20 ± 0.02; p<0.05). These results suggest that reduced inhibitory INH-1 and INH-2 activity, increased PP1C protein level, and reduced PLB phosphorylation are associated with increased PP1 activity in failing hearts. (Mol Cell Biochem 269: 49–57, 2005)     

Cardiac SR-coupled PP1 activity and expression are increased and inhibitor 1 protein expression is decreased in failing hearts

Type 1 protein phosphatase (PP1) is a negative regulator of cardiac function. However, studies on the status and regulation of sarcoplasmic reticulum (SR)-associated PP1 activity in failing hearts are limited. We studied PP1 activity and protein and mRNA expression of the catalytic subunit of PP1 (PP1C) and protein levels of PP1-specific inhibitors [inhibitor 1 (Inh-1) and inhibitor 2 (Inh-2)] in the left ventricular (LV) myocardium of 6 dogs with heart failure (HF; LV ejection fraction, 23 +/- 2%) and 6 normal dogs. In failing LV tissue, PP1 activity values (expressed as pmol 32P. min-1. mg of noncollagen protein-1) in the homogenate, crude membranes, cytosol, and purified SR were increased by 52, 54, 55, and 72%, respectively. Trypsin treatment released PP1 but not type 2A protein phosphatase from the SR. In the supernatant of trypsin-treated SR, PP1 activity was approximately 24% higher in failing hearts than in normal control hearts. A similar increase in protein expression of PP1C was observed in the nontrypsinized SR. Heat-denatured phosphorylated SR inhibited PP1 activity by 30%, which suggests the presence of Inh-1 or -2 or both in the SR. With the use of a specific antibody, both Inh-1 and -2 proteins were found in the SR; the former was decreased by 56% in the failing SR, whereas the latter did not change. These results suggest that protein phosphatase activity bound to the SR is increased and is predominantly type 1. Increased SR-associated PP1 activity in failing hearts appears to be due partly to increased expression of PP1C and partly to reduced levels of Inh-1 but not Inh-2 protein. Thus inhibition of PP1 activity in the SR appears to be a potential therapeutic target for improving LV function in failing hearts, because it may lead to increased SR Ca2+ uptake, which is impaired in failing hearts.     

SERCA2a activity correlates with the force-frequency relationship in human myocardium

The present investigation addresses whether protein expression and function of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) and phospholamban (PLB) correlate in failing and nonfailing human myocardium. SERCA2a activity and protein expression, PLB phosphorylation, and the force-frequency relationship (FFR) have been determined in right atrium (RA) and left ventricle (LV) from nonfailing (NF, n = 12) and terminally failing [dilated cardiomyopathy (DCM), n = 12] human hearts. Only in LV of DCM hearts was SERCA2a activity significantly decreased [maximal turnover rate (V(max)) = 196 +/- 11 and 396 +/- 30 nmol. mg(-1). min(-1) in LV and RA, respectively], whereas protein expression of SERCA2a in the different chambers was unchanged in NF (3.9 +/- 0.3 and 3.2 +/- 0.4 densitometric units in LV and RA, respectively) and DCM hearts (4.8 +/- 0.8 and 3.4 +/- 0.1 densitometric units in LV and RA, respectively). Phosphorylation of PLB was higher in LV than in RA in NF (Ser(16): 180.5 +/- 19.0 vs. 56.8 +/- 6.0 densitometric units; Thr(17): 174.6 +/- 11.2 vs. 37.4 +/- 8.9 densitometric units) and DCM hearts (Ser(16): 132.0 +/- 5.4 vs. 22.4 +/- 3.5 densitometric units; Thr(17): 131.2 +/- 10.9 vs. 9.2 +/- 2.4 densitometric units). SERCA2a function, but not protein expression, correlated well with the functional parameters of the FFR in DCM and NF human hearts. Regulation of SERCA2a function depends on the phosphorylation of PLB at Ser(16) and Thr(17). However, direct SERCA2a regulation might also be affected by an unknown mechanism.     

CaM kinase II and phospholamban contribute to caffeine-induced relaxation of murine gastric fundus smooth muscle

Caffeine has been shown to increase the Ca²⁺ release frequency (Ca²⁺ sparks) from the sarcoplasmic reticulum (SR) through ryanodine-sensitive stores and relax gastric fundus smooth muscle. Increased Ca²⁺ store refilling increases the frequency of Ca²⁺ release events and store refilling is enhanced by CaM kinase II (CaMKII) phosphorylation of phospholamban (PLB). These findings suggest that transient, localized Ca²⁺ release events from the SR may activate CaMKII and contribute to relaxation by enhancing store refilling due to PLB Thr17 phosphorylation. To investigate this possibility, we examined the effects of caffeine on CaMKII, muscle tone, and PLB phosphorylation in murine gastric fundus smooth muscle. Caffeine (1 mM) hyperpolarized and relaxed murine gastric fundus smooth muscle and activated CaMKII. Ryanodine, tetracaine, or cyclopiazonic acid each prevented CaMKII activation and significantly inhibited caffeine-induced relaxation. The large-conductance Ca²⁺-activated K⁺ channel blocker iberiotoxin, but not apamin, partially inhibited caffeine-induced relaxation. Caffeine-induced CaMKII activation increased PLB Thr17, but not PLB Ser16 phosphorylation. 3-Isobutyl-1-methylxanthine increased PLB Ser16 phosphorylation, but not PLB Thr17 phosphorylation. The CaMKII inhibitor KN-93 inhibited caffeine-induced relaxation and PLB Thr17 phosphorylation. These results show that caffeine-induced CaMKII activation and PLB phosphorylation play a role in the relaxation of gastric fundus smooth muscles. Ca²⁺/CaM-dependent protein kinase II     

Ca2+/calmodulin-dependent protein kinase II-dependent remodeling of Ca2+ current in pressure overload heart failure

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity is increased in heart failure (HF), a syndrome characterized by markedly increased risk of arrhythmia. Activation of CaMKII increases peak L-type Ca(2+) current (I(Ca)) and slows I(Ca) inactivation. Whether these events are linked mechanistically is unknown. I(Ca) was recorded in acutely dissociated subepicardial and subendocardial murine left ventricular (LV) myocytes using the whole cell patch clamp method. Pressure overload heart failure was induced by surgical constriction of the thoracic aorta. I(Ca) density was significantly larger in subepicardial myocytes than in subendocardial/myocytes. Similar patterns were observed in the cell surface expression of alpha1c, the channel pore-forming subunit. In failing LV, I(Ca) density was increased proportionately in both cell types, and the time course of I(Ca) inactivation was slowed. This typical pattern of changes suggested a role of CaMKII. Consistent with this, measurements of CaMKII activity revealed a 2-3-fold increase (p < 0.05) in failing LV. To test for a causal link, we measured frequency-dependent I(Ca) facilitation. In HF myocytes, this CaMKII-dependent process could not be induced, suggesting already maximal activation. Internal application of active CaMKII in failing myocytes did not elicit changes in I(Ca). Finally, CaMKII inhibition by internal diffusion of a specific peptide inhibitor reduced I(Ca) density and inactivation time course to similar levels in control and HF myocytes. I(Ca) density manifests a significant transmural gradient, and this gradient is preserved in heart failure. Activation of CaMKII, a known pro-arrhythmic molecule, is a major contributor to I(Ca) remodeling in load-induced heart failure.     

Cardiac calcium dysregulation in mice with chronic kidney disease

Cardiovascular complications are leading causes of morbidity and mortality in patients with chronic kidney disease (CKD). CKD significantly affects cardiac calcium (Ca²⁺) regulation, but the underlying mechanisms are not clear. The present study investigated the modulation of Ca²⁺ homeostasis in CKD mice. Echocardiography revealed impaired fractional shortening (FS) and stroke volume (SV) in CKD mice. Electrocardiography showed that CKD mice exhibited longer QT interval, corrected QT (QTc) prolongation, faster spontaneous activities, shorter action potential duration (APD) and increased ventricle arrhythmogenesis, and ranolazine (10 µmol/L) blocked these effects. Conventional microelectrodes and the Fluo-3 fluorometric ratio techniques indicated that CKD ventricular cardiomyocytes exhibited higher Ca²⁺ decay time, Ca²⁺ sparks, and Ca²⁺ leakage but lower [Ca²⁺]_i transients and sarcoplasmic reticulum Ca²⁺ contents. The CaMKII inhibitor KN93 and ranolazine (RAN; late sodium current inhibitor) reversed the deterioration in Ca²⁺ handling. Western blots revealed that CKD ventricles exhibited higher phosphorylated RyR2 and CaMKII and reduced phosphorylated SERCA2 and SERCA2 and the ratio of PLB-Thr17 to PLB. In conclusions, the modulation of CaMKII, PLB and late Na⁺ current in CKD significantly altered cardiac Ca²⁺ regulation and electrophysiological characteristics. These findings may apply on future clinical therapies.     

Frequency-encoding Thr17 phospholamban phosphorylation is independent of Ser16 phosphorylation in cardiac myocytes

Both Ser(16) and Thr(17) of phospholamban (PLB) are phosphorylated, respectively, by cAMP-dependent protein kinase (PKA) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). PLB phosphorylation relieves cardiac sarcoplasmic reticulum Ca(2+) pump from inhibition by PLB. Previous studies have suggested that phosphorylation of Ser(16) by PKA is a prerequisite for Thr(17) phosphorylation by CaMKII and is essential to the relaxant effect of beta-adrenergic stimulation. To determine the role of Thr(17) PLB phosphorylation, we investigated the dual-site phosphorylation of PLB in isolated adult rat cardiac myocytes in response to beta(1)-adrenergic stimulation or electrical field stimulation (0. 1-3 Hz) or both. A beta(1)-adrenergic agonist, norepinephrine (10(-9)-10(-6) m), in the presence of an alpha(1)-adrenergic antagonist, prazosin (10(-6) m), selectively increases the PKA-dependent phosphorylation of PLB at Ser(16) in quiescent myocytes. In contrast, electrical pacing induces an opposite phosphorylation pattern, selectively enhancing the CaMKII-mediated Thr(17) PLB phosphorylation in a frequency-dependent manner. When combined, electric stimulation (2 Hz) and beta(1)-adrenergic stimulation lead to dual phosphorylation of PLB and exert a synergistic effect on phosphorylation of Thr(17) but not Ser(16). Frequency-dependent Thr(17) phosphorylation is closely correlated with a decrease in 50% relaxation time (t(50)) of cell contraction, which is independent of, but additive to, the relaxant effect of Ser(16) phosphorylation, resulting in hastened contractile relaxation at high stimulation frequencies. Thus, we conclude that in intact cardiac myocytes, phosphorylation of PLB at Thr(17) occurs in the absence of prior Ser(16) phosphorylation, and that frequencydependent Thr(17) PLB phosphorylation may provide an intrinsic mechanism for cardiac myocytes to adapt to a sudden change of heart rate.     

Modulation of late sodium current by Ca2+, calmodulin, and CaMKII in normal and failing dog cardiomyocytes: similarities and differences

Augmented and slowed late Na(+) current (I(NaL)) is implicated in action potential duration variability, early afterdepolarizations, and abnormal Ca(2+) handling in human and canine failing myocardium. Our objective was to study I(NaL) modulation by cytosolic Ca(2+) concentration ([Ca(2+)](i)) in normal and failing ventricular myocytes. Chronic heart failure was produced in 10 dogs by multiple sequential coronary artery microembolizations; 6 normal dogs served as a control. I(NaL) fine structure was measured by whole cell patch clamp in ventricular myocytes and approximated by a sum of fast and slow exponentials produced by burst and late scattered modes of Na(+) channel gating, respectively. I(NaL) greatly enhanced as [Ca(2+)](i) increased from "Ca(2+) free" to 1 microM: its maximum density increased, decay of both exponentials slowed, and the steady-state inactivation (SSI) curve shifted toward more positive potentials. Testing the inhibition of CaMKII and CaM revealed similarities and differences of I(NaL) modulation in failing vs. normal myocytes. Similarities include the following: 1) CaMKII slows I(NaL) decay and decreases the amplitude of fast exponentials, and 2) Ca(2+) shifts SSI rightward. Differences include the following: 1) slowing of I(NaL) by CaMKII is greater, 2) CaM shifts SSI leftward, and 3) Ca(2+) increases the amplitude of slow exponentials. We conclude that Ca(2+)/CaM/CaMKII signaling increases I(NaL) and Na(+) influx in both normal and failing myocytes by slowing inactivation kinetics and shifting SSI. This Na(+) influx provides a novel Ca(2+) positive feedback mechanism (via Na(+)/Ca(2+) exchanger), enhancing contractions at higher beating rates but worsening cardiomyocyte contractile and electrical performance in conditions of poor Ca(2+) handling in heart failure.     

CaM kinase II activation and phospholamban phosphorylation by SNP in murine gastric antrum smooth muscles

Elevations in the intracellular Ca(2+) concentration activate the serine/threonine protein kinase Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). We tested the hypothesis that increased sarco(endo)plasmic reticulum Ca(2+)-ATPase activity by phospholamban (PLB) phosphorylation contributes to smooth muscle relaxation by elevating the sarcoplasmic reticulum (SR) Ca(2+) load and increasing the frequency of Ca(2+) release events from the SR. We have previously shown that caffeine or sodium nitroprusside (SNP) relaxes murine gastric fundus smooth muscles and increases PLB phosphorylation by CaM kinase II. These findings suggest that an increased SR Ca(2+) load increases the frequency of Ca(2+) transients from the SR and results in PLB phosphorylation by CaM kinase II, contributing to caffeine- or SNP-induced relaxation. The aim of the present study was to investigate the effects of SNP on CaM kinase II and PLB phosphorylation in gastric antrum smooth muscles. SNP or 8-bromo-cGMP decreased the basal tone and amplitudes of spontaneous phasic contractions and activated CaM kinase II. SNP-induced relaxation and CaM kinase II activation were blocked by [1,2,4]oxadizolo-[4,3alpha]quinoxaline-1-one (ODQ) and inhibited by cyclopiazonic acid (CPA) or KN-93. SNP also increased PLBSer(16) and PLBThr(17) phosphorylation. Both PLBSer(16) and Thr(17) phosphorylation were ODQ sensitive. However, only PLBThr(17) phosphorylation was inhibited by CPA or KN-93. These results suggest that CaM kinase II activation and PLB phosphorylation participate in the relaxant effect of SNP on murine gastric antrum smooth muscles through a nitric oxide/guanylyl cyclase/cGMP pathway.     

Phosphorylation of phospholamban in ischemia-reperfusion injury: functional role of Thr17 residue

Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser16 by PKA and/or at Thr17 by CaMKII increases the affinity of the SR Ca2+ pump for Ca2+. PLB is therefore, a critical regulator of SR function, myocardial relaxation and myocardial contractility. The present study was undertaken to examine the status of PLB phosphorylation after ischemia and reperfusion and to provide evidence about the possible role of the phosphorylation of Thr17 PLB residue on the recovery of contractility and relaxation after a period of ischemia. Experiments were performed in Langendorff perfused hearts from Wistar rats. Hearts were submitted to a protocol of global normothermic ischemia and reperfusion. The results showed that (1) the phosphorylation of Ser16 and Thr17 residues of PLB increased at the end of the ischemia and the onset of reperfusion, respectively. The increase in Thr17 phosphorylation was associated with a recovery of relaxation to preischemic values. This recovery occurred in spite of the fact that contractility was depressed. (2) The reperfusion-induced increase in Thr17 phosphorylation was dependent on Ca2+ entry to the cardiac cell. This Ca2+ influx would mainly occur by the coupled activation of the Na+ / H+ exchanger and the Na+ / Ca2+ exchanger working in the reverse mode, since phosphorylation of Thr17 was decreased by inhibition of these exchangers and not affected by blockade of the L-type Ca2+ channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased Thr17 phosphorylation. This decrease was associated with an impairment of myocardial relaxation. The present study suggests that the phosphorylation of Thr17 of PLB upon reflow, may favor the full recovery of relaxation after ischemia.     

Role of dual-site phospholamban phosphorylation in intermittent hypoxia-induced cardioprotection against ischemia-reperfusion injury

Cardioprotection by intermittent high-altitude (IHA) hypoxia against ischemia-reperfusion (I/R) injury is associated with Ca(2+) overload reduction. Phospholamban (PLB) phosphorylation relieves cardiac sarcoplasmic reticulum (SR) Ca(2+)-pump ATPase, a critical regulator in intracellular Ca(2+) cycling, from inhibition. To test the hypothesis that IHA hypoxia increases PLB phosphorylation and that such an effect plays a role in cardioprotection, we compared the time-dependent changes in the PLB phosphorylation at Ser(16) (PKA site) and Thr(17) (CaMKII site) in perfused normoxic rat hearts with those in IHA hypoxic rat hearts submitted to 30-min ischemia (I30) followed by 30-min reperfusion (R30). IHA hypoxia improved postischemic contractile recovery, reduced the maximum extent of ischemic contracture, and attenuated I/R-induced depression in Ca(2+)-pump ATPase activity. Although the PLB protein levels remained constant during I/R in both groups, Ser(16) phosphorylation increased at I30 and 1 min of reperfusion (R1) but decreased at R30 in normoxic hearts. IHA hypoxia upregulated the increase further at I30 and R1. Thr(17) phosphorylation decreased at I30, R1, and R30 in normoxic hearts, but IHA hypoxia attenuated the depression at R1 and R30. Moreover, PKA inhibitor H89 abolished IHA hypoxia-induced increase in Ser(16) phosphorylation, Ca(2+)-pump ATPase activity, and the recovery of cardiac performance after ischemia. CaMKII inhibitor KN-93 also abolished the beneficial effects of IHA hypoxia on Thr(17) phosphorylation, Ca(2+)-pump ATPase activity, and the postischemic contractile recovery. These findings indicate that IHA hypoxia mitigates I/R-induced depression in SR Ca(2+)-pump ATPase activity by upregulating dual-site PLB phosphorylation, which may consequently contribute to IHA hypoxia-induced cardioprotection against I/R injury.     

Stress kinase phosphorylation is increased in pacing-induced heart failure in rabbits

In hearts with chronic left ventricular (LV) systolic dysfunction secondary to hypertension or myocardial infarction, MAPK phosphorylation and/or activity are increased. Whether other settings of LV dysfunction not associated with ischemia-reperfusion are also characterized by increased MAPK phosphorylation or activity is unknown. After 3 wk of rapid LV pacing (400 beats/min), eight rabbits displayed clinical signs of heart failure (HF), and echocardiography revealed an increase in LV end-diastolic diameter from 15.6 +/- 0.7 (means +/- SE) to 18.8 +/- 0.7 mm and a reduced shortening fraction from 31 +/- 1to10 +/- 2% (both P < 0.05). Morphological alterations in HF included increased numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cardiomyocytes, extent of fibrosis, and cross-sectional cardiomyocyte area. Total p38 MAPK did not differ between failing and normal hearts (n = 8). However, p38 MAPK phosphorylation [164,488 +/- 29,323 vs. 43,565 +/- 14,817 arbitrary units (AU), P < 0.05, densitometry] and the activities of p38 MAPK-alpha and -beta were increased in failing compared with normal hearts (149,441 +/- 38,381 and 170,430 +/- 32,952 vs. 68,815 +/- 28,984 and 81,788 +/- 22,774 AU, respectively, both P < 0.05). In failing compared with normal hearts, total and phosphorylated JNK46 and JNK54 MAPK were increased, whereas total and phosphorylated ERK MAPK remained unchanged. In pacing-induced HF, p38 and JNK MAPK phosphorylation as well as p38 MAPK activity was increased. Further studies will have to define whether or not chronic specific blockade of MAPK activity can interfere with apoptosis/fibrosis and thereby attenuate the progression of HF.     

Role of conformational sampling of Ser16 and Thr17-phosphorylated phospholamban in interactions with SERCA

Phosphorylation of phospholamban (PLB) at Ser16 and/ or Thr17 is believed to release its inhibitory effect on sarcoplasmic reticulum calcium ATPase. Ser16 phosphorylation of PLB has been suggested to cause a conformational change that alters the interaction between the enzyme and protein. Using computer simulations, the conformational sampling of Ser16 phosphorylated PLB in implicit membrane environment is compared here with the unphosphorylated PLB system to investigate these conformational changes. The results suggest that conformational changes in the cytoplasmic domain of PLB upon phosphorylation at Ser16 increase the likelihood of unfavorable interactions with SERCA in the E2 state prompting a conformational switch of SERCA from E2 to E1. Phosphorylation of PLB at Thr17 on the other hand does not appear to affect interactions with SERCA significantly suggesting that the mechanism of releasing the inhibitory effect is different between Thr17 phosphorylated and Ser16 phosphorylated PLB.     

A single site (Ser16) phosphorylation in phospholamban is sufficient in mediating its maximal cardiac responses to beta -agonists

Phospholamban (PLB) can be phosphorylated at Ser(16) by cyclic AMP-dependent protein kinase and at Thr(17) by Ca(2+)-calmodulin-dependent protein kinase during beta-agonist stimulation. A previous study indicated that mutation of S16A in PLB resulted in lack of Thr(17) phosphorylation and attenuation of the beta-agonist stimulatory effects in perfused mouse hearts. To further delineate the functional interplay between dual-site PLB phosphorylation, we generated transgenic mice expressing the T17A mutant PLB in the cardiac compartment of the null background. Lines expressing similar levels of T17A mutant, S16A mutant, or wild-type PLB in the null background were characterized in parallel. Cardiac myocyte basal mechanics and Ca(2+) kinetics were similar among the three groups. Isoproterenol stimulation was associated with phosphorylation of both Ser(16) and Thr(17) in wild-type PLB and Ser(16) phosphorylation in T17A mutant PLB, whereas there was no detectable phosphorylation of S16A mutant PLB. Phosphorylation of Ser(16) alone in T17A mutant PLB resulted in responses of the mechanical and Ca(2+) kinetic parameters to isoproterenol similar to those in wild-type myocytes, which exhibited dual-site PLB phosphorylation. However, those parameters were significantly attenuated in the S16A mutant myocytes. Thus, Ser(16) in PLB can be phosphorylated independently of Thr(17) in vivo, and phosphorylation of Ser(16) is sufficient for mediating the maximal cardiac responses to beta-adrenergic stimulation.     

Role of dual-site phospholamban phosphorylation in the stunned heart: insights from phospholamban site-specific mutants

Phosphorylation of phospholamban (PLB) at Ser16 (protein kinase A site) and at Thr17 [Ca2+/calmodulin kinase II (CaMKII) site] increases sarcoplasmic reticulum Ca2+ uptake and myocardial contractility and relaxation. In perfused rat hearts submitted to ischemia-reperfusion, we previously showed an ischemia-induced Ser16 phosphorylation that was dependent on beta-adrenergic stimulation and an ischemia and reperfusion-induced Thr17 phosphorylation that was dependent on Ca2+ influx. To elucidate the relationship between these two PLB phosphorylation sites and postischemic mechanical recovery, rat hearts were submitted to ischemia-reperfusion in the absence and presence of the CaMKII inhibitor KN-93 (1 microM) or the beta-adrenergic blocker dl-propranolol (1 microM). KN-93 diminished the reperfusion-induced Thr17 phosphorylation and depressed the recovery of contraction and relaxation after ischemia. dl-Propranolol decreased the ischemia-induced Ser16 phosphorylation but failed to modify the contractile recovery. To obtain further insights into the functional role of the two PLB phosphorylation sites in postischemic mechanical recovery, transgenic mice expressing wild-type PLB (PLB-WT) or PLB mutants in which either Thr17 or Ser16 were replaced by Ala (PLB-T17A and PLB-S16A, respectively) into the PLB-null background were used. Both PLB mutants showed a lower contractile recovery than PLB-WT. However, this recovery was significantly impaired all along reperfusion in PLB-T17A, whereas it was depressed only at the beginning of reperfusion in PLB-S16A. Moreover, the recovery of relaxation was delayed in PLB-T17A, whereas it did not change in PLB-S16A, compared with PLB-WT. These findings indicate that, although both PLB phosphorylation sites are involved in the mechanical recovery after ischemia, Thr17 appears to play a major role.     

Defective Ca(2+) handling proteins regulation during heart failure

Abnormal release of Ca(2+) from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction in heart failure (HF). We previously demonstrated that RyR2 macromolecular complexes from HF rat were significantly more depleted of FK506 binding protein (FKBP12.6). Here we assessed expression of key Ca(2+) handling proteins and measured SR Ca(2+) content in control and HF rat myocytes. Direct measurements of SR Ca(2+) content in permeabilized cardiac myocytes demonstrated that SR luminal [Ca(2+)] is markedly lowered in HF (HF: DeltaF/F(0) = 26.4+/-1.8, n=12; control: DeltaF/F(0) = 49.2+/-2.9, n=10; P<0.01). Furthermore, we demonstrated that the expression of RyR2 associated proteins (including calmodulin, sorcin, calsequestrin, protein phosphatase 1, protein phosphatase 2A), Ca(2+) ATPase (SERCA2a), PLB phosphorylation at Ser16 (PLB-S16), PLB phosphorylation at Thr17 (PLB-T17), L-type Ca(2+) channel (Cav1.2) and Na(+)- Ca(2+) exchanger (NCX) were significantly reduced in rat HF. Our results suggest that systolic SR reduced Ca(2+) release and diastolic SR Ca(2+) leak (due to defective protein-protein interaction between RyR2 and its associated proteins) along with reduced SR Ca(2+) uptake (due to down-regulation of SERCA2a, PLB-S16 and PLB-T17), abnormal Ca(2+) extrusion (due to down-regulation of NCX) and defective Ca(2+) -induced Ca(2+) release (due to down-regulation of Cav1.2) could contribute to HF.