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Differential sensitivity to isoprenaline of troponin I and phospholamban phosphorylation in isolated rat hearts.

ABSTRACT: Phosphorylation of phospholamban (PLB), a membrane-bound 15 kDa protein and troponin I (TNI) was studied in isolated perfused rat hearts by using the back-phosphorylation technique with [32P]ATP catalysed by an excess of exogenous catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, followed by protein separation. This standardized method allows the quantitative detection of protein phosphorylation specifically stimulated by cAMP. In control hearts the extent of specific phosphorylation was equivalent to 3.3 nmol of PLB and 11.0 mumol of TNI per g of cardiac tissue. In hearts freeze-clamped 30 s after exposure to isoprenaline (10 pM-10 microM), there was a dose-dependent decrease in phosphate incorporation in vitro, indicating a phosphorylation of the respective proteins in vivo. A differential sensitivity of TNI and PLB phosphorylation towards the beta-adrenergic agonist and the subsequent increase in tissue cAMP was found, favouring TNI phosphorylation. K0.5 values for isoprenaline were 2.94 +/- 0.04 nM and 4.46 +/- 0.24 nM for PLB and the 15 kDa protein, but 0.13 +/- 0.01 nM for TNI phosphorylation in the intact tissue. At an isoprenaline-induced increase in cAMP less than 3 pmol/mg of protein there was no or only a small increase in PLB phosphorylation, whereas TNI phosphorylation was nearly maximal. By plotting phosphorylation data against changes in contractile parameters a strong correlation was obtained for TNI (r = 0.95), assuming a linear relationship. For PLB a complex relationship is likely to exist. Our data (i) indicate a functional compartmentalization of the cAMP signal cascade and (ii) confirm that phosphorylation of TNI rather than of PLB is related to changes in mechanical myocardial responses.

SUBMITTER: Karczewski P

PROVIDER: S-EPMC1131103 | biostudies-other | 1990 Feb

REPOSITORIES: biostudies-other

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Project description:Ischemic post-conditioning (iPoCo) by coronary re-occlusion/reperfusion during immediate reperfusion after prolonged myocardial ischemia reduces infarct size. Mechanical manipulation of culprit lesions, however, carries the risk of coronary microembolization which may obscure iPoCo's cardioprotection. Pharmacological post-conditioning with exogenous triiodothyronine (T3) could serve as an alternative conditioning strategy. Similar to iPoCo, T3 may activate cardioprotective prosurvival pathways. We aimed to study T3's impact on infarct size and its underlying signal transduction. Hearts were isolated from male Lewis rats (200-380 g), buffer-perfused and subjected to 30 min/120 min global zero-flow ischemia/reperfusion (I/R). In additional hearts, either iPoCo (2 × 30 s/30 s I/R) was performed or T3 (100-500 µg/L) infused at reperfusion. Infarct size was demarcated with triphenyl tetrazolium chloride staining and calculated as percent of ventricular mass. Infarct size was reduced with iPoCo to 16 ± 7% vs. 36 ± 4% with I/R only. The maximum infarct size reduction was observed with 300 µg/L T3 (14 ± 2%). T3 increased the phosphorylation of protein kinase B and mitogen extracellular-regulated-kinase 1/2, both key enzymes of the reperfusion injury salvage kinase (RISK) pathway. Pharmacological RISK blockade (RISK-BL) during reperfusion abrogated T3's cardioprotection (35 ± 10%). Adult ventricular cardiomyocytes were isolated from buffer-perfused rat hearts and exposed to 30 min/5 min hypoxia/reoxygenation (H/R); reoxygenation was initiated without or with T3, respectively, and without or with RISK-BL, respectively. Maximal preservation of viability was observed with 500 µg/L T3 after H/R (27 ± 4% of all cells vs. 5 ± 3% in time-matched controls). Again, RISK-BL abrogated protection (11 ± 3%). Mitochondria were isolated at early reperfusion from buffer-perfused rat hearts without or with iPoCo or 300 µg/L T3, respectively, at reperfusion. T3 improved mitochondrial function (i.e.: increased respiration, adenosine triphosphate production, calcium retention capacity, and decreased reactive oxygen species formation) to a similar extent as iPoCo. T3 at reperfusion reduces infarct size by activation of the RISK pathway. T3's protection is a cardiomyocyte phenomenon and targets mitochondria.

Project description: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.

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Differential sensitivity to isoprenaline of troponin I and phospholamban phosphorylation in isolated rat hearts.

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