scholarly journals Analysis of Mitochondrial Calcium Retention Capacity in Cultured Cells: Permeabilized Cells Versus Isolated Mitochondria

2021 ◽  
Vol 12 ◽  
Author(s):  
Sehwan Jang ◽  
Xavier R. Chapa-Dubocq ◽  
Silvia Fossati ◽  
Sabzali Javadov
Keyword(s):  
Cultured Cells ◽  
Fluorescence Probe ◽  
Permeability Transition ◽  
Colloid Osmotic Pressure ◽  
Inner Mitochondrial Membrane ◽  
Retention Capacity ◽  
Permeabilized Cells ◽  
Calcium Retention ◽  
Isolated Mitochondria ◽  
Calcium Retention Capacity

In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca2+, mitochondria undergo permeability transition (PT) leading to the opening of the non-selective PT pores (PTP) in the inner mitochondrial membrane. Opening of the pores at high conductance allows the passage of ions and solutes <1.5 kD across the membrane, that increases colloid osmotic pressure in the matrix leading to excessive mitochondrial swelling. Calcium retention capacity (CRC) reflects maximum Ca2+ overload of mitochondria that occurs just before PTP opening. Quantification of CRC is important for elucidating the effects of different pathological stimuli and the efficacy of pharmacological agents on the mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the strengths and weaknesses of the CRC technique in isolated cell mitochondria vs. permeabilized cells. The cells were permeabilized by digitonin or saponin, and the Ca2+-sensitive fluorescence probe Calcium Green-5N was used in both preparations. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. Analysis of the CRC in permeabilized cells revealed a higher CRC in the saponin-permeabilized cells in comparison with the digitonin-permeabilized cells. In addition, the mitochondrial CRC in saponin-permeabilized cells was higher than in isolated mitochondria. Altogether, these data demonstrate that the quantification of the mitochondrial CRC in cultured cells permeabilized by saponin has more advantages compared to the isolated mitochondria.

scholarly journals Factors Affecting the Function of the Mitochondrial Membrane Permeability Transition Pore and Their Role in Evaluation of Calcium Retention Capacity Values

2020 ◽  
pp. 491-499
Author(s):  
Z DRAHOTA ◽  
R ENDLICHER ◽  
O KUČERA ◽  
D RYCHTRMOC ◽  
Z ČERVINKOVÁ
Keyword(s):  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Experimental Conditions ◽  
Protein Ratio ◽  
Retention Capacity ◽  
Calcium Retention ◽  
Calcium Retention Capacity

Values of the calcium retention capacity (CRC) of rat liver mitochondria are highly dependent on the experimental conditions used. When increasing amounts of added calcium chloride are used (1.25-10 nmol), the values of the CRC increase 3-fold. When calcium is added in 75 s intervals, the CRC values increase by 30 % compared with 150 s interval additions. CRC values are not dependent on the calcium/protein ratio in the measured sample in our experimental design. We also show that a more detailed evaluation of the fluorescence curves can provide new information about mitochondrial permeability transition pore opening after calcium is added.

scholarly journals Effects of aging on mitochondrial hydrogen peroxide emission and calcium retention capacity in rat heart

2018 ◽  
Vol 14 (6) ◽  
pp. 920-926 ◽  
Author(s):  
Mi-Hyun No ◽  
Jun-Won Heo ◽  
Su-Zi Yoo ◽  
Han-Sam Jo ◽  
Dong-Ho Park ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Rat Heart ◽  
Retention Capacity ◽  
Calcium Retention ◽  
Effects Of Aging ◽  
Calcium Retention Capacity

scholarly journals Chloride channel blocker IAA-94 increases myocardial infarction by reducing calcium retention capacity of the cardiac mitochondria

Life Sciences ◽  
2019 ◽  
Vol 235 ◽  
pp. 116841 ◽  
Author(s):  
Devasena Ponnalagu ◽  
Ahmed Tafsirul Hussain ◽  
Rushi Thanawala ◽  
Jahnavi Meka ◽  
Piotr Bednarczyk ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Chloride Channel ◽  
Channel Blocker ◽  
Cardiac Mitochondria ◽  
Retention Capacity ◽  
Calcium Retention ◽  
Chloride Channel Blocker ◽  
Calcium Retention Capacity

scholarly journals Modification of calcium retention capacity of rat liver mitochondria by phosphate and tert-butyl hydroperoxide

2019 ◽  
pp. 59-65 ◽  
Author(s):  
R. Endlicher ◽  
Z. Drahota ◽  
Z. Červinková
Keyword(s):  
Rat Liver ◽  
Cyclosporine A ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Retention Capacity ◽  
Butyl Hydroperoxide ◽  
Calcium Retention ◽  
Tert Butyl ◽  
Tert Butyl Hydroperoxide ◽  
Calcium Retention Capacity

By determining the calcium retention capacity (CRC) of rat liver mitochondria, we confirmed and extended previous observations describing the activation of mitochondrial swelling by phosphate and tert-butyl hydroperoxide (t-BHP). Using CRC measurements, we showed that both phosphate and t-BHP decrease the extent of calcium accumulation required for the full mitochondrial permeability transition pore (MPTP) opening to 35 % of control values and to only 15 % when both phosphate and t-BHP are present in the medium. When changes in fluorescence were evaluated at higher resolution, we observed that in the presence of cyclosporine A fluorescence values return after each Ca(2+) addition to basal values obtained before the Ca(2+) addition. This indicates that the MPTP remains closed. However, in the absence of cyclosporine A, the basal fluorescence after each Ca(2+) addition continuously increased. This increase was potentiated both by phosphate and t-BHP until the moment when the concentration of intramitochondrial calcium required for the full opening of the MPTP was reached. We conclude that in the absence of cyclosporine A, the MPTP is slowly opened after each Ca(2+) addition and that this rate of opening can be modified by various factors such as the composition of the media and the experimental protocol used.

scholarly journals Low-intensity treadmill exercise protects cognitive impairment by enhancing cerebellar mitochondrial calcium retention capacity in a rat model of chronic cerebral hypoperfusion

2021 ◽  
Vol 17 (5) ◽  
pp. 324-330
Author(s):  
Jae-Min Lee ◽  
Jongmin Park ◽  
Joo-Hee Lee ◽  
Hyo-Bum Kwak ◽  
Mi-Hyun No ◽  
...  
Keyword(s):  
Cognitive Impairment ◽  
Treadmill Exercise ◽  
Neuroprotective Effect ◽  
Cerebral Hypoperfusion ◽  
Mitochondrial Calcium ◽  
Retention Capacity ◽  
Calcium Retention ◽  
Cerebellar Purkinje Cells ◽  
The Brain ◽  
Calcium Retention Capacity

Chronic cerebral hypoperfusion (CCH) is caused by reduced blood flow to the brain representing gradually cognitive impairment. CCH induces mitochondrial dysfunction and neuronal cell death in the brain. Exercise is known to have a neuroprotective effect on brain damage and cognitive dysfunction. This study aimed to clarify the neuroprotective effect of low-intensity treadmill exercise (LITE) by enhancing cerebellar mitochondrial calcium retention capacity in an animal model of CCH. Wistar rats were divided into the sham group, the bilateral common carotid arteries occlusion (BCCAO) group, and the BCCAO and treadmill exercise (BCCAO+Ex) group. BCCAO+Ex group engaged the LITE on a treadmill for 30 min once a day for 8 weeks before the BCCAO surgery to investigate the protective effect of LITE on cognitive impairment. CCH induced by BCCAO resulted in mitochondrial dysfunction in the cerebellum, including impaired calcium homeostasis. CCH also decreased cerebellar Purkinje cells including of calbindin D28k and parvalbumin, resulting in cognitive impairment. The impairment of mitochondrial function, loss of cerebellar Purkinje cells, and cognitive dysfunction ameliorated by exercise. The present study showed that LITE hindered the deficit of spatial working memory and loss of Purkinje cell in the cerebellum induced by CCH. We confirmed that the protective effect of LITE on Purkinje cell by enhanced the mitochondrial calcium retention capacity. We suggest that LITE may protect against cognitive impairment, and further studies are needed to develop the intervention for patients who suffered from CCH.

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