Dataset Information

Chronology of critical events in neonatal rat ventricular myocytes occurring during reperfusion after simulated ischemia.

ABSTRACT: While an ischemic insult poses a lethal danger to myocardial cells, a significant proportion of cardiac myocytes remain viable throughout the ischemic episode and die, paradoxically, only after the blood flow is reinstated. Despite decades of research, the actual chronology of critical events leading to cardiomyocyte death during the reperfusion phase remains poorly understood. Arguably, identification of the pivotal event in this setting is necessary to design effective strategies aimed at salvaging the myocardium after an ischemic attack. Here we used neonatal rat ventricular myocytes (NRVMs) subjected to 20-30 min of simulated ischemia followed by 1 hour of "reperfusion". Using different combinations of spectrally-compatible fluorescent indicators, we analyzed the relative timing of the following events: (1) abnormal increase in cytoplasmic [Ca2+] (TCaCy); (2) abnormal increase in mitochondrial [Ca2+] (TCaMi); (3) loss of mitochondrial inner membrane potential (??m) indicating mitochondrial permeability transitions (TMPT); (4) sacrolemmal permeabilization (SP) to the normally impermeable small fluorophore TO-PRO3 (TSP). In additional experiments we also analyzed the timing of abnormal uptake of Zn2+ into the cytoplasm (TZnCy) relative to TCaCy and TSP. We focused on those NRVMs which survived anoxia, as evidenced by at least 50% recovery of ??m and the absence of detectable SP. In these cells, we found a consistent sequence of critical events in the order, from first to last, of TCaCy, TCaMi, TMPT, TSP. After detecting TCaCy and TCaMi, abrupt switches between 1.1 mM and nominally zero [Ca2+] in the perfusate quickly propagated to the cytoplasmic and mitochondrial [Ca2+]. Depletion of the sarcoplasmic reticulum with ryanodine (5 ?M)/thapsigargin (1 ?M) accelerated all events without changing their order. In the presence of ZnCl2 (10-30 ?M) in the perfusate we found a consistent timing sequence TCaCy < TZn ? TSP. In some cells ZnCl2 interfered with Ca2+ uptake, causing "steps" or "gaps" in the [Ca2+]Cy curve, a phenomenon never observed in the absence of ZnCl2. Together, these findings suggest an evolving permeabilization of NRVM's sarcolemma during reoxygenation, in which the expansion of the pore size determines the timing of critical events, including TMPT.

SUBMITTER: Sciuto KJ

PROVIDER: S-EPMC6366697 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Chronology of critical events in neonatal rat ventricular myocytes occurring during reperfusion after simulated ischemia.

Sciuto Katie J KJ Deng Steven W SW Moreno Alonso A Zaitsev Alexey V AV

PloS one 20190207 2

While an ischemic insult poses a lethal danger to myocardial cells, a significant proportion of cardiac myocytes remain viable throughout the ischemic episode and die, paradoxically, only after the blood flow is reinstated. Despite decades of research, the actual chronology of critical events leading to cardiomyocyte death during the reperfusion phase remains poorly understood. Arguably, identification of the pivotal event in this setting is necessary to design effective strategies aimed at salv ...[more]

PMID: 30730997

Similar Datasets

Project description:Search for new cardioprotective therapies is of great importance since no cardioprotective drugs are available on the market. In line with this need, several natural biomolecules have been extensively tested for their potential cardioprotective effects. Previously, we have shown that biglycan, a member of a diverse group of small leucine-rich proteoglycans, enhanced the expression of cardioprotective genes and decreased ischemia/reperfusion-induced cardiomyocyte death via a TLR-4 dependent mechanism. Therefore, in the present study we aimed to test whether decorin, a small leucine-rich proteoglycan closely related to biglycan, could exert cardiocytoprotection and to reveal possible downstream signaling pathways. Methods: Primary cardiomyocytes isolated from neonatal and adult rat hearts were treated with 0 (Vehicle), 1, 3, 10, 30 and 100 nM decorin as 20 h pretreatment and maintained throughout simulated ischemia and reperfusion (SI/R). In separate experiments, to test the mechanism of decorin-induced cardio protection, 3 nM decorin was applied in combination with inhibitors of known survival pathways, that is, the NOS inhibitor L-NAME, the PKG inhibitor KT-5823 and the TLR-4 inhibitor TAK-242, respectively. mRNA expression changes were measured after SI/R injury. Results: Cell viability of both neonatal and adult cardiomyocytes was significantly decreased due to SI/R injury. Decorin at 1, 3 and 10 nM concentrations significantly increased the survival of both neonatal and adult myocytes after SI/R. At 3nM (the most pronounced protective concentration), it had no effect on apoptotic rate of neonatal cardiac myocytes. No one of the inhibitors of survival pathways (L-NAME, KT-5823, TAK-242) influenced the cardiocytoprotective effect of decorin. MYND-type containing 19 (Zmynd19) and eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1) were significantly upregulated due to the decorin treatment. In conclusion, this is the first demonstration that decorin exerts a direct cardiocytoprotective effect possibly independent of NO-cGMP-PKG and TLR-4 dependent survival signaling.

Project description:Sex differences in responses to myocardial ischemia have been described, but whether cardiomyocyte function is influenced by sex in the setting of ischemia and reperfusion has not been elucidated. This study compared contractions and intracellular Ca(2+) in isolated ventricular myocytes exposed to ischemia and reperfusion. Cells were isolated from anesthetized 3-month-old male and female Fischer 344 rats, paced at 4 Hz (37°C), exposed to simulated ischemia (20 mins) and reperfused. Cell shortening (edge detector) and intracellular Ca(2+) (fura-2) were measured simultaneously. Cell viability was assessed with Trypan blue. Ischemia reduced peak contractions and increased Ca(2+) levels equally in myocytes from both sexes. However, contraction amplitudes were reduced in reperfusion in male myocytes, while contractions recovered to exceed control levels in females (62.6±5.1 vs. 140.1±15.8%; p<0.05). Only 60% of male myocytes excluded trypan blue dye after ischemia and reperfusion, while all female cardiomyocytes excluded the dye (p<0.05). Parallel experiments were conducted in myocytes from ?24-month-old female rats or 5-6-month-old rats that had an ovariectomy at 3-4 weeks of age. Beneficial effects of female sex on myocyte viability and contractile dysfunction in reperfusion were abolished in cells from 24-month-old females. Aged female myocytes also exhibited elevated intracellular Ca(2+) and alternans in ischemia. Cells from ovariectomized rats displayed increased Ca(2+) transients and spontaneous activity in ischemia compared to sham-operated controls. None of the myocytes from ovariectomized rats were viable after 15 minutes of ischemia, while 75% of sham cells remained viable at end of reperfusion (p<0.05). These findings demonstrate that cardiomyocytes from young adult females are more resistant to ischemia and reperfusion injury than cells from males. Age and OVX abolish these beneficial effects and induce Ca(2+) dysregulation at the level of the cardiomyocyte. Thus, beneficial effects of estrogen in ischemia and reperfusion are mediated, in part, by effects on cardiomyocytes.

Dataset Information

Chronology of critical events in neonatal rat ventricular myocytes occurring during reperfusion after simulated ischemia.

Publications

Chronology of critical events in neonatal rat ventricular myocytes occurring during reperfusion after simulated ischemia.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets