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

2003 ◽  
Vol 285 (3) ◽  
pp. H1198-H1205 ◽  
Author(s):  
M. Said ◽  
L. Vittone ◽  
C. Mundiña-Weilenmann ◽  
P. Ferrero ◽  
E. G. Kranias ◽  
...  
Keyword(s):  
Ischemia Reperfusion ◽  
Phosphorylation Sites ◽  
Adrenergic Stimulation ◽  
Rat Hearts ◽  
Adrenergic Blocker ◽  
Perfused Rat Hearts ◽  
A Site ◽  
Contractile Recovery ◽  
Mechanical Recovery

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 β-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 μM) or the β-adrenergic blocker dl-propranolol (1 μM). 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.

Energetics of Ca2+ homeostasis during ischemia–reperfusion on neonatal rat hearts under high-[K+] cardioplegia

2008 ◽  
Vol 86 (12) ◽  
pp. 866-879 ◽  
Author(s):  
Alicia E. Consolini ◽  
Patricia Bonazzola
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Ischemia Reperfusion ◽  
Fibre Length ◽  
Neonatal Rat ◽  
Main Role ◽  
Adult Rats ◽  
Rat Hearts ◽  
High K ◽  
Contractile Recovery

The mechanocalorimetric consequences and mechanisms involved in Ca2+ homeostasis during ischemia–reperfusion (I/R) as well as the protective role of cardioplegic pretreatment with high [K+] (25 mmol/L) and low or near-normal [Ca2+] (0.5 or 2 mmol/L) were evaluated in a model of neonatal rat heart. Beating hearts from 10–12-day-old rats were perfused with Krebs solution (2 mmol/L Ca2+) under both isotonic and isometric conditions. During pretreatment, hearts were exposed for 20 min to either Krebs (control) or cardioplegia (CPG) before 15 min ischemia and 45 min reperfusion while being continuously measured for either contractility or total heat rate (Ht) in a flow calorimeter. Contractile recovery after reperfusion in hearts exposed to ischemia only (control) was higher in the isometric hearts under optimal length (87.9% ± 8.1%) than in the isotonic hearts (57.3% ± 10.6%). This same behavior was found in hearts pretreated with CPG-0.5 mmol/L Ca2+. Ht in controls was reduced from 11.5 ± 0.8 mW/g in the initial beating condition to 1.11 ± 0.33 mW/g during ischemia and was increased to 13.02 ± 0.93 mW/g (113.8% ± 5.0% of preischemic) after reperfusion. Hearts pretreated with CPG-0.5 mmol/L Ca2+ showed the same behavior. However, when extracellular calcium ([Ca]o) was increased to 2 mmol/L under CPG, isotonic hearts, but not isometric hearts, significantly increased the contractile recovery to a maximum of 88.7% ± 10.8% of preischemic levels. Ht was recovered to 92.1% ± 4.3% of preischemic, suggesting that contractile recovery was less energetically expensive after CPG-2 mmol/L Ca2+ than it was in postischemic hearts exposed to control or CPG-0.5 mmol/L Ca2+. The role of the sarcoplasmic reticulum store was evaluated by pretreating hearts with 10 mmol/L caffeine, which reduced contractile recovery only under isometric conditions or after increasing [Ca]o in CPG under isotonic conditions, suggesting that the contribution of the sarcoplasmic reticulum was dependent on the fibre length or the [Ca]o. The inhibition of the reverse mode of the sarcolemmal Na/Ca exchanger (NCX) and the mitochondrial Ca uniporter (CaU) by KB-R7943 (KBR) at 5 µmol/L in CPG-0.5 mmol/L Ca2+ improved contractile recovery of isotonic hearts, whereas it decreased Ht at the start of reperfusion, suggesting that mitochondria could uptake Ca2+ vía the mitochondrial CaU. Neither the positive inotropism nor Ht were changed by inhibiting the mitochondrial NCX with 10 µmol/L clonazepam in CPG-0.5 mmol/L Ca2+ + 5 µmol/L KBR, which suggests that the mitochondrial NCX does not have a role. Finally, the role of the forward mode of the sarcolemmal NCX was evidenced by the fall in contractile recovery with increased Ht when KBR was increased to 20 µmol/L and added to CPG-2 mmol/L Ca2+ + 10 mmol/L caffeine before I/R. Thus the sarcolemmal NCX was essential for removing the diastolic Ca2+ during the periods of CPG and I/R. In summary, Ca2+ homeostasis during I/R of neonatal rat hearts is different from that of adult rats. High-[K+] CPG protected neonatal hearts only under isotonic conditions, at a near-normal [Ca]o, or by exposure to KBR. Mitochondria were able to uptake Ca2+ via the mitochondrial CaU and reduce the Ca2+ available for contractile recovery. Nevertheless, after increasing [Ca]o in CPG, the sarcoplasmic reticulum had a main role in restoring contractility during reperfusion, as it does in adults. Thus, the degree of maturation of the heart must be taken into account to evaluate the effects of CPG and drugs on I/R.

Effects of pyruvate on the energetics of rat ventricles stunned by ischemia–reperfusion

2014 ◽  
Vol 92 (5) ◽  
pp. 386-398 ◽  
Author(s):  
Patricia Bonazzola ◽  
María Inés Ragone ◽  
Alicia E. Consolini
Keyword(s):  
Confocal Microscopy ◽  
Heat Release ◽  
Dynamic Balance ◽  
Ischemia Reperfusion ◽  
Rat Hearts ◽  
High K ◽  
Energetic State ◽  
Contractile Recovery ◽  
Stimulation Of

Pyruvate (Pyr) was proposed as an additive to cold high-K+–low-Ca2+ cardioplegia (CPG) to protect the heart during surgery. We explored whether Pyr and CPG would work synergistically to protect rat hearts from stunning during ischemia–reperfusion (I/R). We measured the heat release and contractility of perfused ventricles during I/R, and the cytosolic and mitochondrial [Ca2+] in cardiomyocytes by confocal microscopy. We found that under cold-CPG (30 °C), 10 mmol·L−1 Pyr reduced the post-ischemic contractile recovery (PICR) as well as muscle economy, when added either before ischemia or during I/R, which was reversed by blockade of UCam. In noncardioplegic hearts, Pyr was cardioprotective when it was present during I/R, more so at 37 °C than at 30 °C, with improved economy. In cardiomyocytes, the addition of Pyr to CPG slightly increased the mitochondrial [Ca2+] but decreased cytosolic [Ca2+]. The results suggest that Pyr only protects hearts from stunning when present before ischemia and during reperfusion, and that it dampens the cardioprotective properties of CPG. The mechanisms underlying such different behavior depend on the dynamic balance between Pyr stimulation of the energetic state and mitochondrial Ca2+ uptake. Our results support the use of Pyr in stunned hearts, but not in cold high-K+ cardioplegia.

Fasudil prevents KATP channel-induced improvement in postischemic functional recovery

2005 ◽  
Vol 288 (6) ◽  
pp. H3011-H3015 ◽  
Author(s):  
Kenya Nishizawa ◽  
Paul E. Wolkowicz ◽  
Tadashi Yamagishi ◽  
Ling-Ling Guo ◽  
Martin M. Pike
Keyword(s):  
Diastolic Pressure ◽  
Rho Kinase ◽  
High Energy ◽  
Left Ventricular ◽  
High Energy Phosphate ◽  
Cellular Processes ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Rock Activity ◽  
Mechanical Recovery

Whereas activation of ATP-dependent potassium (KATP) channels greatly improves postischemic myocardial recovery, the final effector mechanism for KATP channel-induced cardioprotection remains elusive. RhoA is a GTPase that regulates a variety of cellular processes known to be involved with KATP channel cardioprotection. Our goal was to determine whether the activity of a key rhoA effector, rho kinase (ROCK), is required for KATP channel-induced cardioprotection. Four groups of perfused rat hearts were subjected to 36 min of zero-flow ischemia and 44 min of reperfusion with continuous measurements of mechanical function and 31P NMR high-energy phosphate data: 1) untreated, 2) pinacidil (10 μM) to activate KATP channels, 3) fasudil (15 μM) to inhibit ROCK, and 4) both fasudil and pinacidil. Pinacidil significantly improved postischemic mechanical recovery [39 ± 16 vs. 108 ± 4 mmHg left ventricular diastolic pressure (LVDP), untreated and pinacidil, respectively]. Fasudil did not affect reperfusion LVDP (41 ± 13 mmHg) but completely blocked the marked improvement in mechanical recovery that occurred with pinacidil treatment (54 ± 15 mmHg). Substantial attenuation of the postischemic energetic recovery was also observed. These data support the hypothesis that ROCK activity plays a role in KATP channel-induced cardioprotection.

Purinergic Component in the Coronary Vasodilatation to Acetylcholine after Ischemia-Reperfusion in Perfused Rat Hearts

2014 ◽  
Vol 51 (4) ◽  
pp. 283-289 ◽  
Author(s):  
Ángel Luis García-Villalón ◽  
Miriam Granado ◽  
Luis Monge ◽  
Nuria Fernández ◽  
Gonzalo Carreño-Tarragona ◽  
...  
Keyword(s):  
Ischemia Reperfusion ◽  
Rat Hearts ◽  
Coronary Vasodilatation ◽  
Perfused Rat Hearts

scholarly journals Cardiac Ischemia and Ischemia/Reperfusion Cause Wide Proteolysis of the Coronary Endothelial Luminal Membrane: Possible Dysfunctions

2011 ◽  
Vol 5 (1) ◽  
pp. 239-245 ◽  
Author(s):  
Blanca Arroyo-Flores ◽  
Erika Chi-Ahumada ◽  
Erika Briones-Cerecero ◽  
Alma Barajas-Espinosa ◽  
Sandra Perez-Aguilar ◽  
...  
Keyword(s):  
Ischemia Reperfusion ◽  
Biochemical Changes ◽  
Vascular Endothelial ◽  
Luminal Membrane ◽  
Rat Hearts ◽  
Isoelectric Points ◽  
Perfused Rat Hearts ◽  
Hydrolyzing Enzymes ◽  
2D Gel ◽  
G Protein Coupled

Background: Ischemia and ischemia-reperfusion (I/R) are common clinical insults that disrupt the molecular structure of coronary vascular endothelial luminal membrane (VELM) that result in diverse microvasculature dysfunctions. However, the knowledge of the associated biochemical changes is meager. We hypothesized that ischemia and I/R-induced structural and functional VELM alterations result from biochemical changes. First, these changes need to be described and later the mechanisms behind be identified. Methods: During control conditions, in isolated perfused rat hearts VELM proteins were labeled with biotin. The groups of hearts were: control (C), no flow ischemia (I; 25 min), and I/R (I; 25 min, reperfusion 30 min). The biotinylated luminal endothelial membrane proteins in these three different groups were examined by 2-D electrophoresis and identified. But, it must be kept in mind the proteins were biotin-labeled during control. Results: A comparative analysis of the protein profiles under the 3 conditions following 2D gel electrophoresis showed differences in the molecular weight distribution such that MWC > MWI > MWI/R. Similar analysis for isoelectric points (pHi) showed a shift toward more acidic pHi under ischemic conditions. Of 100 % proteins identified during control 66% and 88% changed their MW-pHi during ischemia and I/R respectively. Among these lost proteins there were 9 proteins identified as adhesins and G-protein coupled receptors. General significance: I and I/R insults alter MW-pHi of most luminal glycocalyx proteins due to the activation of nonspecific hydrolizing mechanisms; suspect metalloproteases and glycanases. This makes necessary the identification of hydrolyzing enzymes reponsible of multiple microvascular dysfunctions in order to maintain the integrity of vascular endothelial membrane. VELM must become a target of future therapeutics.

scholarly journals Hydrogen sulfide-mediated cardioprotection against ischemia reperfusion is linked to KATP channel for mitochondrial preservation but not for its distinct preference on interfibrillar mitochondria

2019 ◽  
Vol 14 (2) ◽  
pp. 107-115 ◽  
Author(s):  
Priyadharshini Chandrasekaran ◽  
Sriram Ravindran ◽  
Sri Rahavi Boovarahan ◽  
Gino A. Kurian
Keyword(s):  
Hydrogen Sulfide ◽  
Reperfusion Injury ◽  
Katp Channel ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Rat Hearts ◽  
Significant Difference ◽  
Interfibrillar Mitochondria ◽  
Subsarcolemmal Mitochondria

Hydrogen sulfide has been shown to protect  myocardium against ischemia-reperfusion injury by preserving interfibrillar mitochondria functional activi-ties than subsarcolemmal mitochondria. In this study, the role of the KATP channel in modulating the mitochondrial subpopulations during the cardioprotection mediated by NaSH (H2S donor) was investigated. Isolated rat hearts were treated with mitochondrial KATP channel closer glibenclamide (10 μM)/opener diazoxide (0.8 mM) via Langendorff perfusion apparatus before ischemia-reperfusion. The results showed that NaSH pre-conditioning in presence of glibenclamide significantly improved cardiac recovery without any significant difference between interfibrillar mitochondria and subsarcolemmal mitochondria.  In conclusion, targeting KATP channel may not be good option to target interfibrillar mitochondria/subsarcolemmal mitochondria against ischemia-reperfusion injury.

scholarly journals Real-Time 2-Photon Imaging of Mitochondrial Function in Perfused Rat Hearts Subjected to Ischemia/Reperfusion

Circulation ◽  
2006 ◽  
Vol 114 (14) ◽  
pp. 1497-1503 ◽  
Author(s):  
Madoka Matsumoto-Ida ◽  
Masaharu Akao ◽  
Toshihiro Takeda ◽  
Masashi Kato ◽  
Toru Kita
Keyword(s):  
Real Time ◽  
Mitochondrial Function ◽  
Ischemia Reperfusion ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Photon Imaging

scholarly journals Moderate exercise prevents impaired Ca2+ handling in heart of CO-exposed rat: implication for sensitivity to ischemia-reperfusion

2010 ◽  
Vol 299 (6) ◽  
pp. H2076-H2081 ◽  
Author(s):  
C. Farah ◽  
G. Meyer ◽  
L. André ◽  
J. Boissière ◽  
S. Gayrard ◽  
...  
Keyword(s):  
Exercise Training ◽  
Antioxidant Status ◽  
Ischemia Reperfusion ◽  
Control Group ◽  
Moderate Exercise ◽  
Plasmic Reticulum ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Exercise Induced ◽  
Moderate Exercise Training

Sustained urban carbon monoxide (CO) exposure exacerbates heart vulnerability to ischemia-reperfusion via deleterious effects on the antioxidant status and Ca2+ homeostasis of cardiomyocytes. The aim of this work was to evaluate whether moderate exercise training prevents these effects. Wistar rats were randomly assigned to a control group and to CO groups, living during 4 wk in simulated urban CO pollution (30–100 parts/million, 12 h/day) with (CO-Ex) or sedentary without exercise (CO-Sed). The exercise procedure began 4 wk before CO exposure and was maintained twice a week in standard filtered air during CO exposure. On one set of rats, myocardial ischemia (30 min) and reperfusion (120 min) were performed on isolated perfused rat hearts. On another set of rats, myocardial antioxidant status and Ca2+ handling were evaluated following environmental exposure. As a result, exercise training prevented CO-induced myocardial phenotypical changes. Indeed, exercise induced myocardial antioxidant status recovery in CO-exposed rats, which is accompanied by a normalization of sarco(endo)plasmic reticulum Ca2+-ATPase 2a expression and then of Ca2+ handling. Importantly, in CO-exposed rats, the normalization of cardiomyocyte phenotype with moderate exercise was associated with a restored sensitivity of the myocardium to ischemia-reperfusion. Indeed, CO-Ex rats presented a lower infarct size and a significant decrease of reperfusion arrhythmias compared with their sedentary counterparts. To conclude, moderate exercise, by preventing CO-induced Ca2+ handling and myocardial antioxidant status alterations, reduces heart vulnerability to ischemia-reperfusion.

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