Dataset Information

Isolation, culture, and immunostaining of neonatal rat ventricular myocytes

ABSTRACT: Summary Isolation and culture of ventricular cardiomyocytes from neonatal rats (NRVMs) is a powerful model to study neonatal cardiac development, cell cycle regulation, and cardiac physiology and pathology in vitro. Here, we present our modified enzymatic digestion protocol followed by two-step discontinuous Percoll gradient centrifugation to isolate a high yield of viable ventricular cardiomyocytes from neonatal rats. Finally, here we describe an immunostaining protocol for cytosolic and nuclear staining of NRVMs. For complete details on the use and execution of this protocol, please refer to Pereira et al. (2020). Graphical abstract Highlights • Isolate a high yield of viable ventricular cardiomyocytes from neonatal rats (NRVMs)• NRVMs can be maintained in culture for several days• Enzymatic digestion of cardiac tissue and Percoll gradient separation• Detailed subsection of immunofluorescence for nuclear and cytosolic staining Isolation and culture of ventricular cardiomyocytes from neonatal rats (NRVMs) is a powerful model to study neonatal cardiac development, cell cycle regulation, and cardiac physiology and pathology in vitro. Here, we present our modified enzymatic digestion protocol followed by two-step discontinuous Percoll gradient centrifugation to isolate a high yield of viable ventricular cardiomyocytes from neonatal rats. Finally, here we describe an immunostaining protocol for cytosolic and nuclear staining of NRVMs.

SUBMITTER: Pereira A

PROVIDER: S-EPMC8599495 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description: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 (ΔΨm) 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 ΔΨm 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 μM)/thapsigargin (1 μM) accelerated all events without changing their order. In the presence of ZnCl2 (10-30 μM) 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.

Dataset Information

Isolation, culture, and immunostaining of neonatal rat ventricular myocytes

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets