Ablation of phospholamban rescues reperfusion arrhythmias but exacerbates myocardium infarction in hearts with Ca2+/calmodulin kinase II constitutive phosphorylation of ryanodine receptors.

AIMS: Abnormal Ca2+ release from the sarcoplasmic reticulum (SR), associated with Ca2+-calmodulin kinase II (CaMKII)-dependent phosphorylation of RyR2 at Ser2814, has consistently been linked to arrhythmogenesis and ischaemia/reperfusion (I/R)-induced cell death. In contrast, the role played by SR Ca2+ uptake under these stress conditions remains controversial. We tested the hypothesis that an increase in SR Ca2+ uptake is able to attenuate reperfusion arrhythmias and cardiac injury elicited by increased RyR2-Ser2814 phosphorylation. METHODS AND RESULTS: We used WT mice, which have been previously shown to exhibit a transient increase in RyR2-Ser2814 phosphorylation at the onset of reperfusion; mice with constitutive pseudo-phosphorylation of RyR2 at Ser2814 (S2814D) to exacerbate CaMKII-dependent reperfusion arrhythmias and cardiac damage, and phospholamban (PLN)-deficient-S2814D knock-in (SDKO) mice resulting from crossbreeding S2814D with phospholamban knockout deficient (PLNKO) mice. At baseline, S2814D and SDKO mice had structurally normal hearts. Moreover none of the strains were arrhythmic before ischaemia. Upon cardiac I/R, WT, and S2814D hearts exhibited abundant arrhythmias that were prevented by PLN ablation. In contrast, PLN ablation increased infarct size compared with WT and S2814D hearts. Mechanistically, the enhanced SR Ca2+ sequestration evoked by PLN ablation in SDKO hearts prevented arrhythmogenic events upon reperfusion by fragmenting SR Ca2+ waves into non-propagated and non-arrhythmogenic events (mini-waves). Conversely, the increase in SR Ca2+ sequestration did not reduce but rather exacerbated I/R-induced SR Ca2+ leak, as well as mitochondrial alterations, which were greatly avoided by inhibition of RyR2. These results indicate that the increase in SR Ca2+ uptake is ineffective in preventing the enhanced SR Ca2+ leak of PLN ablated myocytes from either entering into nearby mitochondria and/or activating additional CaMKII pathways, contributing to cardiac damage. CONCLUSION: Our results demonstrate that increasing SR Ca2+ uptake by PLN ablation can prevent the arrhythmic events triggered by CaMKII-dependent phosphorylation of RyR2-induced SR Ca2+ leak. These findings underscore the benefits of increasing SERCA2a activity in the face of SR Ca2+ triggered arrhythmias. However, enhanced SERCA2a cannot prevent but rather exacerbates I/R cardiac injury.

Unbalance Between Sarcoplasmic Reticulum Ca2 + Uptake and Release: A First Step Toward Ca2 + Triggered Arrhythmias and Cardiac Damage.

The present review focusses on the regulation and interplay of cardiac SR Ca2+ handling proteins involved in SR Ca2+ uptake and release, i.e., SERCa2/PLN and RyR2. Both RyR2 and SERCA2a/PLN are highly regulated by post-translational modifications and/or different partners' proteins. These control mechanisms guarantee a precise equilibrium between SR Ca2+ reuptake and release. The review then discusses how disruption of this balance alters SR Ca2+ handling and may constitute a first step toward cardiac damage and malignant arrhythmias. In the last part of the review, this concept is exemplified in different cardiac diseases, like prediabetic and diabetic cardiomyopathy, digitalis intoxication and ischemia-reperfusion injury.

Underlying mechanism of the contractile dysfunction in atrophied ventricular myocytes from a murine model of hypothyroidism.

Hypothyroidism (Hypo) is a risk factor for cardiovascular diseases, including heart failure. Hypo rapidly induces Ca2+ mishandling and contractile dysfunction (CD), as well as atrophy and ventricular myocytes (VM) remodeling. Hypo decreases SERCA-to-phospholamban ratio (SERCA/PLB), and thereby contributes to CD. Nevertheless, detailed spatial and temporal Ca2+ cycling characterization in VM is missing, and contribution of other structural and functional changes to the mechanism underlying Ca2+ mishandling and CD, as transverse tubules (T-T) remodeling, mitochondrial density (Dmit) and energy availability, is unclear. Therefore, in a rat model of Hypo, we aimed to characterize systolic and diastolic Ca2+ signaling, T-T remodeling, Dmit, citrate synthase (CS) activity and high-energy phosphate metabolites (ATP and phosphocreatine). We confirmed a decrease in SERCA/PLB (59%), which slowed SERCA activity (48%), reduced SR Ca2+ (19%) and blunted Ca2+ transient amplitude (41%). Moreover, assessing the rate of SR Ca2+ release (dRel/dt), we found that early and maximum dRel/dt decreased, and this correlated with staggered Ca2+ transients. However, dRel/dt persisted during Ca2+ transient relaxation due to abundant late Ca2+ sparks. Isoproterenol significantly up-regulated systolic Ca2+ cycling. T-T were unchanged, hence, cannot explain staggered Ca2+ transients and altered dRel/dt. Therefore, we suggest that these might be caused by RyR2 clusters desynchronization, due to diminished Ca2+-dependent sensitivity of RyR2, which also caused a decrease in diastolic SR Ca2+ leak. Furthermore, Dmit was unchanged and CS activity slightly decreased (14%), however, the ratio phosphocreatine/ATP did not change, therefore, energy deficiency cannot account for Ca2+ and contractility dysregulation. We conclude that decreased SR Ca2+, due to slower SERCA, disrupts systolic RyR2 synchronization, and this underlies CD.

Effects of exercise training on excitation-contraction coupling, calcium dynamics and protein expression in the heart of the Neotropical fish Brycon amazonicus.

Matrinxã (Brycon amazonicus) is a great swimming performance teleost fish from the Amazon basin. However, the possible cardiac adaptations of this ability are still unknown. Therefore, the aim of the present work was to investigate the effects of prolonged exercise (EX group - 60days under 0.4BL·s-1) on ventricular contractility by (i) in-vitro analysis of contractility comparing the relative roles of sodium/calcium exchanger (NCX) and sarcoplasmic reticulum (SR) in the excitation-contraction (E-C) coupling and (ii) molecular analysis of NCX, sarcoplasmic reticulum Ca2+ ATPase (SERCA2) and phospholamban (PLB) expression and quantification. The exercise training significantly improved twitch tension, cardiac pumping capacity and the contraction rate when compared to controls (CT). Inhibition of the NCX function, replacing Na+ by Li+ in the physiological solutions, diminished cardiac contractility in the EX group, reduced all analyzed parameters under both high and low stimulation frequencies. The SR blockage, using 10µM ryanodine, caused ~50% tension reduction in CT at most analyzed frequencies while in EX, reductions (34-54%) were only found at higher frequencies. SR inhibition also decreased contraction and relaxation rates in both groups. Additionally, higher post-rest contraction values were recorded for EX, indicating an increase in SR Ca2+ loading. Higher NCX and PLB expression rates and lower SERCA2 rates were found in EX. Our data indicate that matrinxã presents a modulation in E-C coupling after exercise-training, enhancing the SR function under higher frequencies. This was the first study to functionally analyze the effects of swimming-induced exercise on fish cardiac E-C coupling.

Molecular Changes in Children with Heart Failure Undergoing Left Ventricular Assist Device Therapy.

OBJECTIVE: To determine whether left ventricular assist device (LVAD) treatment in children with heart failure would result in the modification of molecular pathways involved in heart failure pathophysiology. STUDY DESIGN: Forty-seven explanted hearts from children were studied (16 nonfailing control, 20 failing, and 11 failing post-LVAD implantation [F-LVAD]). Protein expression and phosphorylation states were determined by receptor binding assays and Western blots. mRNA expression was measured with real-time quantitative polymerase chain reaction. To evaluate for interactions and identify correlations, 2-way ANOVA and regression analysis were performed. RESULTS: Treatment with LVAD resulted in recovery of total ß-adrenergic receptor expression and ß1-adrenergic receptor (ß1-AR) in failing hearts to normal levels (ß-adrenergic receptor expression : 67.2 ± 11.5 fmol/mg failing vs 99.5 ± 27.7 fmol/mg nonfailing, 104 ± 38.7 fmol/mg F-LVAD, P ≤ .01; ß1-AR: 52.2 ± 10.3 fmol/mg failing vs 83.0 ± 23 fmol/mg non-failing, 76.5 ± 32.1 fmol/mg F-LVAD P ≤ .03). The high levels of G protein-coupled receptor kinase-2 were returned to nonfailing levels after LVAD treatment (5.6 ± 9.0 failing vs 1.0 ± 0.493 nonfailing, 1.0 ± 1.3 F-LVAD). Interestingly, ß2-adrenergic receptor expression was significantly greater in F-LVAD (27.5 ± 12; P < .005) hearts compared with nonfailing (16.4 ± 6.1) and failing (15.1 ± 4.2) hearts. Phospholamban phosphorylation at serine 16 was significantly greater in F-LVAD (7.7 ± 11.7) hearts compared with nonfailing (1.0 ± 1.2, P = .02) and failing (0.8 ± 1.0, P = .01) hearts. Also, atrial natriuretic factor (0.6 ± 0.8) and brain natriuretic peptide (0.1 ± 0.1) expression in F-LVAD was significantly lower compared with failing hearts (2.8 ± 3.6, P = .01 and 0.6 ± 0.7, P = .02). CONCLUSION: LVAD treatment in children with heart failure results in reversal of several pathologic myocellular processes, and G protein-coupled receptor kinase-2 may regulate ß1-AR but not ß2-adrenergic receptor expression in children with heart failure.

Myocardial Response to Milrinone in Single Right Ventricle Heart Disease.

OBJECTIVES: Empiric treatment with milrinone, a phosphodiesterase (PDE) 3 inhibitor, has become increasingly common in patients with single ventricle heart disease of right ventricular (RV) morphology (SRV); our objective was to characterize the myocardial response to PDE3 inhibition (PDE3i) in the pediatric population with SRV. STUDY DESIGN: Cyclic adenosine monophosphate levels, PDE activity, and phosphorylated phospholamban (PLN) were determined in explanted human ventricular myocardium from nonfailing pediatric donors (n = 10) and pediatric patients transplanted secondary to SRV. Subjects with SRV were further classified by PDE3i treatment (n = 13 with PDE3i and n = 12 without PDE3i). RESULTS: In comparison with nonfailing RV myocardium (n = 8), cyclic adenosine monophosphate levels are lower in patients with SRV treated with PDE3i (n = 12, P = .021). Chronic PDE3i does not alter total PDE or PDE3 activity in SRV myocardium. Compared with nonfailing RV myocardium, SRV myocardium (both with and without PDE3i) demonstrates equivalent phosphorylated PLN at the protein kinase A phosphorylation site. CONCLUSIONS: As evidenced by preserved phosphorylated PLN, the molecular adaptation associated with SRV differs significantly from that demonstrated in pediatric heart failure because of dilated cardiomyopathy. These alterations support a pathophysiologically distinct mechanism of heart failure in pediatric patients with SRV, which has direct implications regarding the presumed response to PDE3i treatment in this population.

Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50 percent diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50 percent food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

Phosphorylation of phospholamban in ischemia-reperfusion injury: Functional role of Thr(17) residue.

Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser(16) by PKA and/or at Thr(17) by CaMKII increases the affinity of the SR Ca(2+) pump for Ca(2+). 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 Thr(17) 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 Ser(16) and Thr(17) residues of PLB increased at the end of the ischemia and the onset of reperfusion, respectively. The increase in Thr(17) 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 Thr(17) phosphorylation was dependent on Ca(2+) entry to the cardiac cell. This Ca(2+) influx would mainly occur by the coupled activation of the Na(+)/H(+) exchanger and the Na(+)/Ca(2+) exchanger working in the reverse mode, since phosphorylation of Thr(17) was decreased by inhibition of these exchangers and not affected by blockade of the L-type Ca(2+) channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased Thr(17) phosphorylation. This decrease was associated with an impairment of myocardial relaxation. The present study suggests that the phosphorylation of Thr(17) of PLB upon reflow, may favor the full recovery of relaxation after ischemia. (Mol Cell Biochem 263: 131-136, 2004).