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Updated: Aug 8, 2026
Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay
Published on: May 1, 2018
CRYAB and HSPB2 deficiency increases myocyte mitochondrial permeability transition and mitochondrial calcium uptake
Toshie Kadono1, Xiu Quan Zhang, Sathya Srinivasan
1Department of Internal Medicine, Division of Cardiology, University of Utah Health Sciences Center, 50 North Medical Drive, Salt Lake City, UT 84132, USA.
Abstract:
Double knockout (DKO) of the small heat shock proteins CRYAB and HSPB2 increases necrosis and apoptosis induced by ischemia/reperfusion (I/R) in vitro, but the mechanisms involved are unknown. We examined [Ca2+]i during metabolic inhibition (MI) changes in [Ca2+]m induced by exposure to elevated [Ca2+]i, and whether mitochondria in isolated DKO ventricular myocytes (VM) are more susceptible than wild type (WT) to induction of the mitochondrial permeability transition (MPT). The rise in [Ca2+]i in DKO myocytes during metabolic inhibition (MI) was less than in WT, and ouabain caused a greater increase in [Ca2+]m in DKO than in WT. These findings suggested that Ca2+ uptake was increased in mitochondria in DKO myocytes. Measurements of Rhod 2 fluorescence during exposure of permeabilized VM to 1000 nM [Ca2+] for 5 min confirmed that DKO myocytes have enhanced mitochondrial Ca2+ uptake, and this difference between DKO and WT myocyte mitochondria was eliminated by inhibition of NO synthesis. MPT was induced more readily by ouabain, PAO, or TMRM in DKO myocytes than in WT. Thus, Ca2+ uptake by mitochondria is increased in DKO VM by a NO-dependent mechanism. This can predispose to the development of MPT, and increased VM injury during I/R. These findings indicate an important role of CRYAB and/or HSPB2 in mitochondrial function.
Insights
Small heat shock proteins CRYAB and HSPB2 protect heart cells. Their absence increases cell injury during ischemia/reperfusion by enhancing mitochondrial calcium uptake via a nitric oxide-dependent pathway.
Area of Science:
- Cardiovascular Biology
- Mitochondrial Physiology
- Cellular Stress Response
Background:
- Small heat shock proteins CRYAB and HSPB2 play a role in cellular protection.
- Their absence (double knockout, DKO) exacerbates injury from ischemia/reperfusion (I/R).
- The underlying mechanisms for this increased susceptibility remain unclear.
Purpose of the Study:
- To investigate the role of CRYAB and HSPB2 in regulating mitochondrial calcium handling and susceptibility to mitochondrial permeability transition (MPT).
- To elucidate the mechanisms by which DKO of CRYAB and HSPB2 affects cardiomyocyte (VM) injury during metabolic inhibition (MI) and I/R.
Main Methods:
- Utilized isolated ventricular myocytes (VM) from wild-type (WT) and DKO mice.
- Measured intracellular calcium ([Ca2+]i) and mitochondrial calcium ([Ca2+]m) dynamics during metabolic inhibition and calcium challenges.
- Assessed MPT induction using pharmacological agents and measured mitochondrial calcium uptake via Rhod 2 fluorescence.
- Investigated the role of nitric oxide (NO) synthesis inhibition.
Main Results:
- DKO myocytes exhibited reduced [Ca2+]i rise during MI but enhanced [Ca2+]m accumulation.
- Mitochondria in DKO myocytes demonstrated increased calcium uptake, a process dependent on nitric oxide (NO) synthesis.
- DKO myocytes were more susceptible to MPT induction by various stimuli compared to WT.
Conclusions:
- Increased mitochondrial calcium uptake in DKO VM, mediated by a NO-dependent mechanism, enhances susceptibility to MPT.
- This heightened MPT predisposition contributes to increased ventricular myocyte injury during I/R.
- CRYAB and/or HSPB2 are crucial for maintaining normal mitochondrial function and protecting against I/R-induced injury.
