Methods in cardiomyocyte isolation, culture, and gene transfer

Louch, William E.; Sheehan, Katherine A.; Wolska, Beata M.

doi:10.1016/j.yjmcc.2011.06.012

Cited by 413 publications

(413 citation statements)

References 118 publications

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“…Interestingly, the inhibition of GSK3β by SB21 did not modify the caffeineinduced Ca 2+ transfer from SR/ER to mitochondria (P = NS, Figure 2c), suggesting that GSK3β has a specific action on IP 3 R channels with no effect on RyRs in adult cardiomyocytes. As adult mice cardiomyocytes are refractory to transfection using conventional methods and that they can only be maintained in culture for short periods before they dedifferentiate, 23 the analysis on adult cardiomyocytes was complemented by a number of assays on H9c2 cells to define the role of GSK3β in the regulation of Ca 2+ transfer between SR/ER and mitochondria. Similar to adult cardiomyocytes, rhod-2 showed a specific mitochondrial staining in H9c2 cells with an overlap coefficient averaging 0.87 ± 0.02 when compared with Mitotracker Green (Supplementary Figure S3A).…”

Section: Resultsmentioning

confidence: 99%

The SR/ER-mitochondria calcium crosstalk is regulated by GSK3β during reperfusion injury

Gomez¹,

Thiebaut²,

Paillard³

et al. 2015

Cell Death Differ

103

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Glycogen synthase kinase-3β (GSK3β) is a multifunctional kinase whose inhibition is known to limit myocardial ischemiareperfusion injury. However, the mechanism mediating this beneficial effect still remains unclear. Mitochondria and sarco/ endoplasmic reticulum (SR/ER) are key players in cell death signaling. Their involvement in myocardial ischemia-reperfusion injury has gained recognition recently, but the underlying mechanisms are not yet well understood. We questioned here whether GSK3β might have a role in the Ca 2+ transfer from SR/ER to mitochondria at reperfusion. We showed that a fraction of GSK3β protein is localized to the SR/ER and mitochondria-associated ER membranes (MAMs) in the heart, and that GSK3β specifically interacted with the inositol 1,4,5-trisphosphate receptors (IP 3 Rs) Ca 2+ channeling complex in MAMs. We demonstrated that both pharmacological and genetic inhibition of GSK3β decreased protein interaction of IP 3 R with the Ca 2+ channeling complex, impaired SR/ER Ca 2+ release and reduced the histamine-stimulated Ca 2+ exchange between SR/ER and mitochondria in cardiomyocytes. During hypoxia reoxygenation, cell death is associated with an increase of GSK3β activity and IP 3 R phosphorylation, which leads to enhanced transfer of Ca 2+ from SR/ER to mitochondria. Inhibition of GSK3β at reperfusion reduced both IP 3 R phosphorylation and SR/ER Ca 2+ release, which consequently diminished both cytosolic and mitochondrial Ca 2+ concentrations, as well as sensitivity to apoptosis. We conclude that inhibition of GSK3β at reperfusion diminishes Ca 2+ leak from IP 3 R at MAMs in the heart, which limits both cytosolic and mitochondrial Ca 2+ overload and subsequent cell death. Glycogen synthase kinase-3 (GSK3) was originally identified as a phosphorylating kinase for glycogen synthase. 1,2 It has two isoforms, α and β, that possess strong homology in their kinase domains with, however, distinct functions. 3 GSK3 is constitutively active but it can be inhibited by phosphorylation on serine 21 (Ser21) for GSK3α and Ser9 for GSK3β. 4 In the heart, GSK3β has several important roles in cardiac hypertrophy 5 and ischemia-reperfusion (IR) injury. 6 Accumulating evidence indicates that phospho-Ser9-GSK3β-mediated cytoprotection is achieved by an increased threshold for permeability transition pore (PTP) opening. [6][7][8][9] The mechanism by which GSK3β delays PTP opening still remains unclear. It has been reported that GSK3β could interact with ANT at the inner mitochondrial membrane in the heart 9 and/or to phosphorylate voltage-dependent anion channel (VDAC) and cyclophilin D (CypD) in cancer cells. 10,11 GSK3β also has other proposed mechanisms of action, including a poorly characterized role in calcium (Ca 2+ ) homeostasis regulation 12 and protein-protein interactions, 9 as well as functions in different subcellular fractions such as the nucleus, cytosol and mitochondria. 13 Reperfusion is the most powerful intervention to salvage ischemic myocardium. However, it can also paradoxically lead to ca...

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Section: Resultsmentioning

confidence: 99%

The SR/ER-mitochondria calcium crosstalk is regulated by GSK3β during reperfusion injury

Gomez¹,

Thiebaut²,

Paillard³

et al. 2015

Cell Death Differ

103

View full text Add to dashboard Cite

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“…Neonatal rat cardiac myocytes and fibroblasts were isolated as previously reported 22. The senescent cardiomyocyte model was established by culturing neonatal rat cardiac myocytes under hypoxia (1% O 2 , 5% CO 2 , 94% N 2 ) for 10 hours, manifesting as significantly increased SA‐β‐gal activity.…”

Section: Methodsmentioning

confidence: 99%

Postinfarction Hearts Are Protected by Premature Senescent Cardiomyocytes Via GATA4‐Dependent CCN1 Secretion

Cui

Xue

Yang

et al. 2018

JAHA

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BackgroundStress‐induced cell premature senescence participates in a variety of tissue and organ remodeling by secreting such proteins as proinflammatory cytokines, chemokines, and growth factors. However, the role of cardiomyocyte senescence in heart remodeling after acute myocardial infarction has not been thoroughly elucidated to date. Therefore, we sought to clarify the impact of premature myocardial senescence on postinfarction heart function.Methods and ResultsSenescence markers, including p16INK 4a, p21CIP 1/ WAF 1, and SA‐β‐gal staining, were analyzed in several heart disease models by immunostaining. Both postinfarction mouse hearts and ischemic human myocardium demonstrated increased senescence markers. Additionally, senescence‐related secretory phenotype was activated after acute myocardial infarction, which upregulated senescence‐related secretory phenotype factors, including CCN family member 1 (CCN1), interleukin‐1α, tumor necrosis factor α, and monocyte chemoattractant protein‐1. In vivo, a tail vein injection of AAV9‐Gata4‐shRNA significantly attenuated senescence‐related secretory phenotype secretion and aggravated postinfarction heart dysfunction. Furthermore, among activated senescence‐related secretory phenotype factors, CCN1 administration reduced myofibroblast viability in vitro and rescued the deleterious effect of AAV9‐Gata4‐shRNA in vivo.ConclusionsMyocardial premature senescence was observed in the ischemic hearts and improved postinfarction heart function, partly through the GATA‐binding factor 4‐CCN1 pathway.

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“…Ventricular myocytes were isolated from 8‐week‐old WT and REEP5 ‐KO littermates using a previously described method 14. Each cellular experiment used cardiomyocytes isolated from 5 sex‐matched rats per genotype.…”

Section: Methodsmentioning

confidence: 99%

REEP5 (Receptor Accessory Protein 5) Acts as a Sarcoplasmic Reticulum Membrane Sculptor to Modulate Cardiac Function

Yao

Xie

et al. 2018

JAHA

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BackgroundHeart failure is a complex syndrome characterized by cardiac contractile impairment with high mortality. Defective intracellular Ca2+ homeostasis is the central cause under this scenario and tightly links to ultrastructural rearrangements of sarcolemmal transverse tubules and the sarcoplasmic reticulum (SR); however, the modulators of the SR architecture remain unknown. The SR has been thought to be a specialized endoplasmic reticulum membrane system. Receptor accessory proteins (REEPs)/DP1/Yop1p are responsible for shaping high‐curvature endoplasmic reticulum tubules. This study aimed to determine the role of REEPs in SR membrane shaping and thus cardiac function.Methods and ResultsWe identified REEP5 (receptor accessory protein 5) as more highly expressed than other REEP members in adult rat ventricular myocardium, and it was downregulated in the failing hearts. Targeted inactivation of REEP5 in rats specially deformed the cardiac SR membrane without affecting transverse tubules, and this was visualized by focused ion beam scanning electron microscopy–based 3‐dimensional reconstruction. Accordingly, simultaneous recordings of depolarization‐induced Ca2+ currents and Ca2+ transients in REEP5‐null cardiomyocytes revealed normal L‐type Ca2+ channel currents but a depressed SR Ca2+ release. Consequently, the excitation–contraction coupling gain of cardiomyocytes and consequent cardiac contractility were compromised. REEP5 deficiency did not alter the expression of major proteins involved in Ca2+ handling in the heart.Conclusions REEP5 modulates cardiac function by shaping the SR. REEP5 defect deforms the SR architecture to depress cardiac contractility. REEP5‐dependent SR shaping might have potential as a therapeutic target for heart failure.

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Methods in cardiomyocyte isolation, culture, and gene transfer

Cited by 413 publications

References 118 publications

The SR/ER-mitochondria calcium crosstalk is regulated by GSK3β during reperfusion injury

The SR/ER-mitochondria calcium crosstalk is regulated by GSK3β during reperfusion injury

Postinfarction Hearts Are Protected by Premature Senescent Cardiomyocytes Via GATA4‐Dependent CCN1 Secretion

REEP5 (Receptor Accessory Protein 5) Acts as a Sarcoplasmic Reticulum Membrane Sculptor to Modulate Cardiac Function

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