Enhanced cell volume regulation: a key protective mechanism of ischemic preconditioning in rabbit ventricular myocytes

Video microscopy was used to study the regulation of cell volume in isolated rabbit ventricular myocytes. Myocytes rapidly (less than or equal to 2 min) swelled and shrank in hyposmotic and hyperosmotic solutions, respectively, and this initial volume response was maintained without a regulatory volume decrease or increase for 20 min. Relative cell volumes (normalized to isosmotic solution, 1T) were as follows: 1.41 +/- 0.01 in 0.6T, 1.20 +/- 0.04 in 0.8T, 0.71 +/- 0.04 in 1.8T, and 0.57 +/- 0.03 in 2.6T. These volume changes were significantly less than expected if all of the measured volume was osmotically active water. Changes in width and thickness were significantly greater than changes in cell length. The idea that cotransport contributes to cell volume regulation was tested by inhibiting Na(+)-K(+)-2Cl- cotransport with bumetanide (BUM) and Na(+)-Cl- cotransport with chlorothiazide (CTZ). Under isotonic conditions, a 10-min exposure to BUM (1 microM), CTZ (100 microM), or BUM (10 microM) plus CTZ (100 microM) decreased relative cell volume to 0.87 +/- 0.01, 0.86 +/- 0.02, and 0.82 +/- 0.04, respectively. BUM plus CTZ also modified the response to osmotic stress. Swelling in 2.6T medium was 76% greater and shrinkage in 0.6T medium was 29% less than in the absence of diuretics. In contrast to the rapid effects of diuretics, inhibition of the Na(+)-K+ pump with 10 microM ouabain for 20 min did not affect cell volume in 1T solution. Nevertheless, ouabain decreased swelling in 0.6T medium by 52% and increased shrinkage in 1.8T medium by 34%. These data suggest that under isotonic conditions Na(+)-K(+)-2Cl- and Na(+)-Cl- cotransport are critical in establishing cell volume, but osmoregulation can compensate for Na(+)-K+ pump inhibition for at least 20 min. Under anisotonic conditions, the Na(+)-K+ pump and Na(+)-K(+)-2Cl- and/or Na(+)-Cl- cotransport are important in myocyte volume regulation.

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Swelling-activated Gd3+-sensitive Cation Current and Cell Volume Regulation in Rabbit Ventricular Myocytes

The Journal of General Physiology ◽

10.1085/jgp.110.3.297 ◽

1997 ◽

Vol 110 (3) ◽

pp. 297-312 ◽

Cited By ~ 51

Author(s):

Henry F. Clemo ◽

Clive M. Baumgarten

Keyword(s):

Cell Volume ◽

Volume Change ◽

Volume Regulation ◽

Ventricular Myocytes ◽

Cell Volume Regulation ◽

Hill Coefficient ◽

Physiological Processes ◽

Cation Current ◽

Voltage Dependent ◽

Rabbit Ventricular Myocytes

The role of swelling-activated currents in cell volume regulation is unclear. Currents elicited by swelling rabbit ventricular myocytes in solutions with 0.6–0.9× normal osmolarity were studied using amphotericin perforated patch clamp techniques, and cell volume was examined concurrently by digital video microscopy. Graded swelling caused graded activation of an inwardly rectifying, time-independent cation current (ICir,swell) that was reversibly blocked by Gd3+, but ICir,swell was not detected in isotonic or hypertonic media. This current was not related to IK1 because it was insensitive to Ba2+. The PK/PNa ratio for ICir,swell was 5.9 ± 0.3, implying that inward current is largely Na+ under physiological conditions. Increasing bath K+ increased gCir,swell but decreased rectification. Gd3+ block was fitted with a K0.5 of 1.7 ± 0.3 μM and Hill coefficient, n, of 1.7 ± 0.4. Exposure to Gd3+ also reduced hypotonic swelling by up to ∼30%, and block of current preceded the volume change by ∼1 min. Gd3+-induced cell shrinkage was proportional to ICir,swell when ICir,swell was varied by graded swelling or Gd3+ concentration and was voltage dependent, reflecting the voltage dependence of ICir,swell. Integrating the blocked ion flux and calculating the resulting change in osmolarity suggested that ICir,swell was sufficient to explain the majority of the volume change at –80 mV. In addition, swelling activated an outwardly rectifying Cl− current, ICl,swell. This current was absent after Cl− replacement, reversed at ECl, and was blocked by 1 mM 9-anthracene carboxylic acid. Block of ICl,swell provoked a 28% increase in swelling in hypotonic media. Thus, both cation and anion swelling-activated currents modulated the volume of ventricular myocytes. Besides its effects on cell volume, ICir,swell is expected to cause diastolic depolarization. Activation of ICir,swell also is likely to affect contraction and other physiological processes in myocytes.

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Cell-Volume Regulation by Swelling-Activated Chloride Current in Guinea-Pig Ventricular Myocytes

The Japanese Journal of Physiology ◽

10.2170/jjphysiol.54.31 ◽

2004 ◽

Vol 54 (1) ◽

pp. 31-38 ◽

Cited By ~ 14

Author(s):

Shintaro Yamamoto ◽

Keiko Ishihara ◽

Tsuguhisa Ehara ◽

Takao Shioya

Keyword(s):

Guinea Pig ◽

Cell Volume ◽

Volume Regulation ◽

Ventricular Myocytes ◽

Cell Volume Regulation ◽

Chloride Current

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Remote Ischemic Preconditioning Cardio-protection via Enhanced Cell Volume Regulation Requires Activation of Swelling-Activated Chloride (Icl- Swell) Channels

Cardiovascular Pharmacology Open Access ◽

10.4172/2329-6607.1000117 ◽

2014 ◽

Vol 03 (01) ◽

Author(s):

Gregory J Wilson

Keyword(s):

Cell Volume ◽

Ischemic Preconditioning ◽

Volume Regulation ◽

Cell Volume Regulation ◽

Remote Ischemic Preconditioning ◽

Cardio Protection

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Enhanced cell volume regulation: a key mechanism in local and remote ischemic preconditioning

AJP Cell Physiology ◽

10.1152/ajpcell.00259.2013 ◽

2014 ◽

Vol 306 (12) ◽

pp. C1191-C1199 ◽

Cited By ~ 9

Author(s):

Roberto J. Diaz ◽

Kordan Harvey ◽

Azadeh Boloorchi ◽

Taneya Hossain ◽

Alina Hinek ◽

...

Keyword(s):

Cell Volume ◽

Ischemic Preconditioning ◽

Volume Regulation ◽

Regulatory Volume Decrease ◽

Cell Volume Regulation ◽

Dominant Negative ◽

Cell Swelling ◽

Blue Staining ◽

Common Mechanism ◽

Channel Blockade

We have previously shown that ischemic preconditioning (IPC) protection against necrosis in whole hearts and in both fresh and cultured cardiomyocytes, as well as the improved regulatory volume decrease to hypoosmotic swelling in cardiomyocytes, is abrogated through Cl− channel blockade, pointing to a role for enhanced cell volume regulation in IPC. To further define this cardioprotective mechanism, cultured rabbit ventricular cardiomyocytes were preconditioned either by 10-min simulated ischemia (SI) followed by 10-min simulated reperfusion (SR), by 10-min exposure/10-min washout of remote IPC (rIPC) plasma dialysate (from rabbits subjected to repetitive limb ischemia), or by adenoviral transfection with the constitutively active PKC-ε gene. These interventions were done before cardiomyocytes were subjected to either 60- or 75-min SI/60-min SR to assess cell necrosis (by trypan blue staining), 30-min SI to assess ischemic cell swelling, or 30-min hypoosmotic (200 mosM) stress to assess cell volume regulation. Necrosis after SI/SR and both SI- and hypoosmotic stress-induced swelling was reduced in preconditioned cardiomyocytes compared with control cardiomyocytes (neither preconditioned nor transfected). These effects on necrosis and cell swelling were blocked by either Cl− channel blockade or dominant negative knockdown of inwardly rectifying K+ channels with adenoviruses, suggesting that Cl− and K+ movements across the sarcolemma are critical for cell volume regulation and, thereby, cell survival under hypoxic/ischemic conditions. Our results define enhanced cell volume regulation as a key common mechanism of cardioprotection by preconditioning in cardiomyocytes.

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