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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.Ischemia reduced peak contractions and increased Ca(2+) levels equally in myocytes from both sexes.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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OVX promoted spontaneous contractions and Ca2+ transients in ischemia when compared to sham-operated controls.Cells were paced with trains of 20 pulses, delivered at a frequency of 4 Hz, followed by a 2.5 seconds delay to observe spontaneous activity. A. Representative examples of stimulated and spontaneous Ca2+ transients (top) and contractions (bottom) recorded in an OVX female myocyte after 5 minutes of exposure to ischemia. The first three beats are stimulated beats and the spontaneous responses are shown by the arrows. B. Spontaneous activity (Ca2+ transients and contractions) occurred in 71.4% of OVX cells. In contrast, spontaneous activity was not observed in myocytes from sham-operated animals. The * denotes significantly different from values in sham-operated females (p<0.05; n = 7 sham cells and 7 OVX cells).
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pone-0038425-g007: OVX promoted spontaneous contractions and Ca2+ transients in ischemia when compared to sham-operated controls.Cells were paced with trains of 20 pulses, delivered at a frequency of 4 Hz, followed by a 2.5 seconds delay to observe spontaneous activity. A. Representative examples of stimulated and spontaneous Ca2+ transients (top) and contractions (bottom) recorded in an OVX female myocyte after 5 minutes of exposure to ischemia. The first three beats are stimulated beats and the spontaneous responses are shown by the arrows. B. Spontaneous activity (Ca2+ transients and contractions) occurred in 71.4% of OVX cells. In contrast, spontaneous activity was not observed in myocytes from sham-operated animals. The * denotes significantly different from values in sham-operated females (p<0.05; n = 7 sham cells and 7 OVX cells).

Mentions: To determine whether OVX modified ischemia and reperfusion injury, responses were compared in myocytes from sham and OVX rats. Note that none of the OVX myocytes were viable after 15 minutes of ischemia (Figure 6A–D, arrows), so effects late in the protocol were not available. While contractions declined in ischemia in sham controls, contractions remained large in the initial ischemic period in OVX cells (Figure 6A). Ca2+ transients also increased in ischemia in OVX cells, but not in sham controls (Figure 6B). However, the increase in diastolic Ca2+ levels in ischemia was similar in sham and OVX myocytes (Figure 6C) and diastolic length did not differ between the two groups (Figure 6D). Figure 6E shows that OVX dramatically reduced the ability of cardiomyocytes to tolerate ischemia. While most sham myocytes remained viable following exposure to ischemia and reperfusion, none of the OVX myocytes remained viable beyond 15 minutes of ischemia and this difference was statistically significant (p<0.05). Furthermore, 71% of OVX myocytes showed spontaneous Ca2+ release and contractions in ischemia, as shown in the example in Figure 7A. By contrast, spontaneous activity did not occur in myocytes from sham-operated controls (Figure 7B). These results show that OVX disrupted Ca2+ homeostasis and abolished the beneficial effects of female sex on cell viability in individual cardiomyocytes.


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)

OVX promoted spontaneous contractions and Ca2+ transients in ischemia when compared to sham-operated controls.Cells were paced with trains of 20 pulses, delivered at a frequency of 4 Hz, followed by a 2.5 seconds delay to observe spontaneous activity. A. Representative examples of stimulated and spontaneous Ca2+ transients (top) and contractions (bottom) recorded in an OVX female myocyte after 5 minutes of exposure to ischemia. The first three beats are stimulated beats and the spontaneous responses are shown by the arrows. B. Spontaneous activity (Ca2+ transients and contractions) occurred in 71.4% of OVX cells. In contrast, spontaneous activity was not observed in myocytes from sham-operated animals. The * denotes significantly different from values in sham-operated females (p<0.05; n = 7 sham cells and 7 OVX cells).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368849&req=5

pone-0038425-g007: OVX promoted spontaneous contractions and Ca2+ transients in ischemia when compared to sham-operated controls.Cells were paced with trains of 20 pulses, delivered at a frequency of 4 Hz, followed by a 2.5 seconds delay to observe spontaneous activity. A. Representative examples of stimulated and spontaneous Ca2+ transients (top) and contractions (bottom) recorded in an OVX female myocyte after 5 minutes of exposure to ischemia. The first three beats are stimulated beats and the spontaneous responses are shown by the arrows. B. Spontaneous activity (Ca2+ transients and contractions) occurred in 71.4% of OVX cells. In contrast, spontaneous activity was not observed in myocytes from sham-operated animals. The * denotes significantly different from values in sham-operated females (p<0.05; n = 7 sham cells and 7 OVX cells).
Mentions: To determine whether OVX modified ischemia and reperfusion injury, responses were compared in myocytes from sham and OVX rats. Note that none of the OVX myocytes were viable after 15 minutes of ischemia (Figure 6A–D, arrows), so effects late in the protocol were not available. While contractions declined in ischemia in sham controls, contractions remained large in the initial ischemic period in OVX cells (Figure 6A). Ca2+ transients also increased in ischemia in OVX cells, but not in sham controls (Figure 6B). However, the increase in diastolic Ca2+ levels in ischemia was similar in sham and OVX myocytes (Figure 6C) and diastolic length did not differ between the two groups (Figure 6D). Figure 6E shows that OVX dramatically reduced the ability of cardiomyocytes to tolerate ischemia. While most sham myocytes remained viable following exposure to ischemia and reperfusion, none of the OVX myocytes remained viable beyond 15 minutes of ischemia and this difference was statistically significant (p<0.05). Furthermore, 71% of OVX myocytes showed spontaneous Ca2+ release and contractions in ischemia, as shown in the example in Figure 7A. By contrast, spontaneous activity did not occur in myocytes from sham-operated controls (Figure 7B). These results show that OVX disrupted Ca2+ homeostasis and abolished the beneficial effects of female sex on cell viability in individual cardiomyocytes.

Bottom Line: Cell shortening (edge detector) and intracellular Ca(2+) (fura-2) were measured simultaneously.Ischemia reduced peak contractions and increased Ca(2+) levels equally in myocytes from both sexes.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