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Age and ovariectomy abolish beneficial effects of female sex on rat ventricular myocytes exposed to simulated ischemia and reperfusion.

Ross JL, Howlett SE - PLoS ONE (2012)

Bottom Line: Cell shortening (edge detector) and intracellular Ca(2+) (fura-2) were measured simultaneously.However, contraction amplitudes were reduced in reperfusion in male myocytes, while contractions recovered to exceed control levels in females (62.6±5.1 vs. 140.1±15.8%; p<0.05).Age and OVX abolish these beneficial effects and induce Ca(2+) dysregulation at the level of the cardiomyocyte.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.

ABSTRACT
Sex differences in responses to myocardial ischemia have been described, but whether cardiomyocyte function is influenced by sex in the setting of ischemia and reperfusion has not been elucidated. This study compared contractions and intracellular Ca(2+) in isolated ventricular myocytes exposed to ischemia and reperfusion. Cells were isolated from anesthetized 3-month-old male and female Fischer 344 rats, paced at 4 Hz (37°C), exposed to simulated ischemia (20 mins) and reperfused. Cell shortening (edge detector) and intracellular Ca(2+) (fura-2) were measured simultaneously. Cell viability was assessed with Trypan blue. Ischemia reduced peak contractions and increased Ca(2+) levels equally in myocytes from both sexes. However, contraction amplitudes were reduced in reperfusion in male myocytes, while contractions recovered to exceed control levels in females (62.6±5.1 vs. 140.1±15.8%; p<0.05). Only 60% of male myocytes excluded trypan blue dye after ischemia and reperfusion, while all female cardiomyocytes excluded the dye (p<0.05). Parallel experiments were conducted in myocytes from ∼24-month-old female rats or 5-6-month-old rats that had an ovariectomy at 3-4 weeks of age. Beneficial effects of female sex on myocyte viability and contractile dysfunction in reperfusion were abolished in cells from 24-month-old females. Aged female myocytes also exhibited elevated intracellular Ca(2+) and alternans in ischemia. Cells from ovariectomized rats displayed increased Ca(2+) transients and spontaneous activity in ischemia compared to sham-operated controls. None of the myocytes from ovariectomized rats were viable after 15 minutes of ischemia, while 75% of sham cells remained viable at end of reperfusion (p<0.05). These findings demonstrate that cardiomyocytes from young adult females are more resistant to ischemia and reperfusion injury than cells from males. Age and OVX abolish these beneficial effects and induce Ca(2+) dysregulation at the level of the cardiomyocyte. Thus, beneficial effects of estrogen in ischemia and reperfusion are mediated, in part, by effects on cardiomyocytes.

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Ischemia inhibited contractions, enhanced diastolic Ca2+ loading, and promoted post-ischemic contractile dysfunction in myocytes from young adult male rats.Cells were paced at a frequency of 4 Hz for 15 minutes in normal Tyrode’s buffer, exposed to simulated ischemia for 20 minutes and reperfused with normal Tyrode’s for 30 minutes (filled squares). Time control cells were paced for the same length of time in normal Tyrode’s buffer only (open squares). A. Representative examples of Ca2+ transients (top) and contractions (bottom) recorded prior to ischemia, after 5 minutes of exposure to ischemia and after 20 minutes of reperfusion. B. Mean (± SEM) peak contractions recorded at 5 minute intervals throughout the experimental protocol. C. Mean amplitudes of Ca2+ transients recorded during these experiments. Mean levels of diastolic Ca2+ (D) and diastolic cell length (E) recorded throughout the protocol. In all cases, responses were normalized to values recorded after 15 minutes of stimulation, prior to exposure to ischemia. The * denotes significantly different from time control (p<0.05; n = 5 time control cells and 12 cells exposed to ischemia and reperfusion).
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pone-0038425-g001: Ischemia inhibited contractions, enhanced diastolic Ca2+ loading, and promoted post-ischemic contractile dysfunction in myocytes from young adult male rats.Cells were paced at a frequency of 4 Hz for 15 minutes in normal Tyrode’s buffer, exposed to simulated ischemia for 20 minutes and reperfused with normal Tyrode’s for 30 minutes (filled squares). Time control cells were paced for the same length of time in normal Tyrode’s buffer only (open squares). A. Representative examples of Ca2+ transients (top) and contractions (bottom) recorded prior to ischemia, after 5 minutes of exposure to ischemia and after 20 minutes of reperfusion. B. Mean (± SEM) peak contractions recorded at 5 minute intervals throughout the experimental protocol. C. Mean amplitudes of Ca2+ transients recorded during these experiments. Mean levels of diastolic Ca2+ (D) and diastolic cell length (E) recorded throughout the protocol. In all cases, responses were normalized to values recorded after 15 minutes of stimulation, prior to exposure to ischemia. The * denotes significantly different from time control (p<0.05; n = 5 time control cells and 12 cells exposed to ischemia and reperfusion).

Mentions: To determine whether responses of individual cardiomyocytes to myocardial ischemia were influenced by the sex of the animal, contractions and underlying Ca2+ transients were compared throughout ischemia and reperfusion in myocytes from young adult male and female rats. Figure 1A shows Ca2+ transients (top) and contractions (bottom) recorded from a male myocyte at selected time points throughout an experiment. Figure 1B–C shows mean peak contractions and Ca2+ transients recorded from male myocytes during ischemia and reperfusion compared to responses in time controls. Data were normalized to values recorded after 15 minutes of pacing at 4 Hz to facilitate comparisons between groups. Contractions were essentially abolished by ischemia but recovered with an overshoot immediately upon reperfusion (Figure 1B). However, peak contractions remained smaller than time controls with continued reperfusion (Figure 1B). In contrast, ischemia and reperfusion had no effect on Ca2+ transients throughout the experiment (Figure 1C). Ischemia caused a marked increase in diastolic Ca2+ and this recovered upon reperfusion (Figure 1D). Reperfusion also was associated with a modest degree of hypercontracture (Figure 1E). There were no signs of spontaneous activity in ischemia or reperfusion in this group (not shown). However, Trypan blue staining revealed that 38% of male myocytes were trypan blue positive by the end of the reperfusion period. Thus, male myocytes exposed to ischemia exhibited increased diastolic Ca2+, along with post-ischemic contractile dysfunction (stunning) and reduced viability in reperfusion.


Age and ovariectomy abolish beneficial effects of female sex on rat ventricular myocytes exposed to simulated ischemia and reperfusion.

Ross JL, Howlett SE - PLoS ONE (2012)

Ischemia inhibited contractions, enhanced diastolic Ca2+ loading, and promoted post-ischemic contractile dysfunction in myocytes from young adult male rats.Cells were paced at a frequency of 4 Hz for 15 minutes in normal Tyrode’s buffer, exposed to simulated ischemia for 20 minutes and reperfused with normal Tyrode’s for 30 minutes (filled squares). Time control cells were paced for the same length of time in normal Tyrode’s buffer only (open squares). A. Representative examples of Ca2+ transients (top) and contractions (bottom) recorded prior to ischemia, after 5 minutes of exposure to ischemia and after 20 minutes of reperfusion. B. Mean (± SEM) peak contractions recorded at 5 minute intervals throughout the experimental protocol. C. Mean amplitudes of Ca2+ transients recorded during these experiments. Mean levels of diastolic Ca2+ (D) and diastolic cell length (E) recorded throughout the protocol. In all cases, responses were normalized to values recorded after 15 minutes of stimulation, prior to exposure to ischemia. The * denotes significantly different from time control (p<0.05; n = 5 time control cells and 12 cells exposed to ischemia and reperfusion).
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Related In: Results  -  Collection

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pone-0038425-g001: Ischemia inhibited contractions, enhanced diastolic Ca2+ loading, and promoted post-ischemic contractile dysfunction in myocytes from young adult male rats.Cells were paced at a frequency of 4 Hz for 15 minutes in normal Tyrode’s buffer, exposed to simulated ischemia for 20 minutes and reperfused with normal Tyrode’s for 30 minutes (filled squares). Time control cells were paced for the same length of time in normal Tyrode’s buffer only (open squares). A. Representative examples of Ca2+ transients (top) and contractions (bottom) recorded prior to ischemia, after 5 minutes of exposure to ischemia and after 20 minutes of reperfusion. B. Mean (± SEM) peak contractions recorded at 5 minute intervals throughout the experimental protocol. C. Mean amplitudes of Ca2+ transients recorded during these experiments. Mean levels of diastolic Ca2+ (D) and diastolic cell length (E) recorded throughout the protocol. In all cases, responses were normalized to values recorded after 15 minutes of stimulation, prior to exposure to ischemia. The * denotes significantly different from time control (p<0.05; n = 5 time control cells and 12 cells exposed to ischemia and reperfusion).
Mentions: To determine whether responses of individual cardiomyocytes to myocardial ischemia were influenced by the sex of the animal, contractions and underlying Ca2+ transients were compared throughout ischemia and reperfusion in myocytes from young adult male and female rats. Figure 1A shows Ca2+ transients (top) and contractions (bottom) recorded from a male myocyte at selected time points throughout an experiment. Figure 1B–C shows mean peak contractions and Ca2+ transients recorded from male myocytes during ischemia and reperfusion compared to responses in time controls. Data were normalized to values recorded after 15 minutes of pacing at 4 Hz to facilitate comparisons between groups. Contractions were essentially abolished by ischemia but recovered with an overshoot immediately upon reperfusion (Figure 1B). However, peak contractions remained smaller than time controls with continued reperfusion (Figure 1B). In contrast, ischemia and reperfusion had no effect on Ca2+ transients throughout the experiment (Figure 1C). Ischemia caused a marked increase in diastolic Ca2+ and this recovered upon reperfusion (Figure 1D). Reperfusion also was associated with a modest degree of hypercontracture (Figure 1E). There were no signs of spontaneous activity in ischemia or reperfusion in this group (not shown). However, Trypan blue staining revealed that 38% of male myocytes were trypan blue positive by the end of the reperfusion period. Thus, male myocytes exposed to ischemia exhibited increased diastolic Ca2+, along with post-ischemic contractile dysfunction (stunning) and reduced viability in reperfusion.

Bottom Line: Cell shortening (edge detector) and intracellular Ca(2+) (fura-2) were measured simultaneously.However, contraction amplitudes were reduced in reperfusion in male myocytes, while contractions recovered to exceed control levels in females (62.6±5.1 vs. 140.1±15.8%; p<0.05).Age and OVX abolish these beneficial effects and induce Ca(2+) dysregulation at the level of the cardiomyocyte.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.

ABSTRACT
Sex differences in responses to myocardial ischemia have been described, but whether cardiomyocyte function is influenced by sex in the setting of ischemia and reperfusion has not been elucidated. This study compared contractions and intracellular Ca(2+) in isolated ventricular myocytes exposed to ischemia and reperfusion. Cells were isolated from anesthetized 3-month-old male and female Fischer 344 rats, paced at 4 Hz (37°C), exposed to simulated ischemia (20 mins) and reperfused. Cell shortening (edge detector) and intracellular Ca(2+) (fura-2) were measured simultaneously. Cell viability was assessed with Trypan blue. Ischemia reduced peak contractions and increased Ca(2+) levels equally in myocytes from both sexes. However, contraction amplitudes were reduced in reperfusion in male myocytes, while contractions recovered to exceed control levels in females (62.6±5.1 vs. 140.1±15.8%; p<0.05). Only 60% of male myocytes excluded trypan blue dye after ischemia and reperfusion, while all female cardiomyocytes excluded the dye (p<0.05). Parallel experiments were conducted in myocytes from ∼24-month-old female rats or 5-6-month-old rats that had an ovariectomy at 3-4 weeks of age. Beneficial effects of female sex on myocyte viability and contractile dysfunction in reperfusion were abolished in cells from 24-month-old females. Aged female myocytes also exhibited elevated intracellular Ca(2+) and alternans in ischemia. Cells from ovariectomized rats displayed increased Ca(2+) transients and spontaneous activity in ischemia compared to sham-operated controls. None of the myocytes from ovariectomized rats were viable after 15 minutes of ischemia, while 75% of sham cells remained viable at end of reperfusion (p<0.05). These findings demonstrate that cardiomyocytes from young adult females are more resistant to ischemia and reperfusion injury than cells from males. Age and OVX abolish these beneficial effects and induce Ca(2+) dysregulation at the level of the cardiomyocyte. Thus, beneficial effects of estrogen in ischemia and reperfusion are mediated, in part, by effects on cardiomyocytes.

Show MeSH
Related in: MedlinePlus