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Jan 24, 2014 - cycle. The typical mouse estrous cycle lasts 4 to 5 days and consists of four stages, proestrus, estrus, metestrus, and di- estrus, which can be ...
Am J Physiol Heart Circ Physiol 306: H938 –H953, 2014. First published January 24, 2014; doi:10.1152/ajpheart.00730.2013.

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Cardiac contraction, calcium transients, and myofilament calcium sensitivity fluctuate with the estrous cycle in young adult female mice Jennifer K. MacDonald,1 W. Glen Pyle,2 Cristine J. Reitz,2 and Susan E. Howlett1,3 1

Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada; 2Cardiovascular Research Group, Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada; and 3Department of Medicine (Geriatric Medicine), Dalhousie University, Halifax, Nova Scotia, Canada Submitted 23 September 2013; accepted in final form 6 January 2014

MacDonald JK, Pyle WG, Reitz CJ, Howlett SE. Cardiac contraction, calcium transients, and myofilament calcium sensitivity fluctuate with the estrous cycle in young adult female mice. Am J Physiol Heart Circ Physiol 306: H938 –H953, 2014. First published January 24, 2014; doi:10.1152/ajpheart.00730.2013.—This study established conditions to induce regular estrous cycles in female C57BL/6J mice and investigated the impact of the estrous cycle on contractions, Ca2⫹ transients, and underlying cardiac excitation-contraction (EC)-coupling mechanisms. Daily vaginal smears from group-housed virgin female mice were stained to distinguish estrous stage (proestrus, estrus, metestrus, diestrus). Ventricular myocytes were isolated from anesthetized mice. Contractions and Ca2⫹ transients were measured simultaneously (4 Hz, 37°C). Interestingly, mice did not exhibit regular cycles unless they were exposed to male pheromones in bedding added to their cages. Field-stimulated myocytes from mice in estrus had larger contractions (⬃2-fold increase), larger Ca2⫹ transients (⬃1.11-fold increase), and longer action potentials (⬎2-fold increase) compared with other stages. Larger contractions and Ca2⫹ transients were not observed in estrus myocytes voltage-clamped with shorter action potentials. Voltage-clamp experiments also demonstrated that estrous stage had no effect on Ca2⫹ current, EC-coupling gain, diastolic Ca2⫹, sarcoplasmic reticulum (SR) Ca2⫹ content, or fractional release. Although contractions were largest in estrus, myofilament Ca2⫹ sensitivity was lowest (EC50 values ⬃1.15-fold higher) in conjunction with increased phosphorylation of myosin binding protein C in estrus. Contractions were enhanced in ventricular myocytes from mice in estrus because action potential prolongation increased SR Ca2⫹ release. These findings demonstrate that cyclical changes in reproductive hormones associated with the estrous cycle can influence myocardial electrical and contractile function and modify Ca2⫹ homeostasis. However, such changes are unlikely to occur in female mice housed in groups under conventional conditions, since these mice do not exhibit regular estrous cycles. sex; gender; sex hormones

there are important differences in cardiac physiology and pathophysiology between men and women (44, 53, 86). This should not be surprising, since cardiac myocytes possess receptors for all major sex steroid hormones, including estrogen, testosterone, and progesterone (24, 25, 47, 65). Indeed, there is growing evidence that sex IT IS WELL ESTABLISHED THAT

Address for reprint requests and other correspondence: S. E. Howlett, Dept. of Pharmacology, 5850 College St., PO Box 15000, Sir Charles Tupper Medical Bldg., Dalhousie Univ., Halifax, Nova Scotia, Canada B3H 4R2 (e-mail: [email protected]). H938

steroid hormones can modulate the electrical and contractile function of the heart, in part, through actions on the myocytes themselves (61). Despite this knowledge, the majority of experimental studies of cardiac function under normal and pathological conditions have used only male animals. This bias against the use of female animals arises, in part, because of concerns that varying levels of reproductive hormones may increase data heterogeneity and confound results (3). In laboratory rodents, cyclical changes in the levels of circulating reproductive hormones give rise to the estrous cycle. The typical mouse estrous cycle lasts 4 to 5 days and consists of four stages, proestrus, estrus, metestrus, and diestrus, which can be distinguished by vaginal cytology (8, 9, 82). During the follicular phase of the estrous cycle, circulating levels of estradiol rise throughout proestrus and peak just before ovulation at estrus (22, 77, 82). Follicle-stimulating hormone and luteinising hormone levels also peak before ovulation (59). Progesterone levels rise during metestrus and diestrus, which correspond to the luteal phase of the estrous cycle, and then decline (22, 77, 82). Interestingly, female pheromones are known to suppress the estrous cycle when female mice are housed in groups (40, 41, 81). Therefore, regular estrous cycles may not occur in female mice housed in conventional animal care facilities, unless they are induced to cycle through exposure to male pheromones, a phenomenon known as the Whitten effect (80). Although information is limited, some studies in rodents exhibiting regular estrous cycles suggest that fluctuating levels of reproductive hormones may modify myocardial function. Myocardial performance is not affected by estrous stage under basal conditions in isolated, perfused rat hearts (6, 84). However, hearts from rats in proestrus are resistant to myocardial injury induced by either hypoxia (6) or trauma-hemorrhage (84). Furthermore, hearts from rats in proestrus are slower to develop ventricular fibrillation following coronary ligation when compared with rats at other stages (27). Also, evidence shows that the estrous cycle affects cardiac function at the level of the individual cardiomyocyte. Myocytes from mice in estrus exhibit lower K⫹ current densities (Ito,f, IK,slow, but not IKir) and longer action potential durations (APD) when compared with mice in diestrus, although APD was not examined in the other estrous stages (67). Prolongation of the APD in estrus compared with diestrus has also been observed in the guinea pig model (31). These observations suggest that changes in

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reproductive hormone levels associated with the estrous cycle can affect cardiac excitation at the level of the myocyte. Cardiac excitation triggers sarcoplasmic reticulum (SR) Ca2⫹ release and initiates contraction, a process known as excitation-contraction (EC) coupling (5). The estrous cycle has been shown to affect cardiomyocyte excitation and thus may also modify Ca2⫹ homeostasis and contraction, although this has not yet been investigated. This is important, since abnormal Ca2⫹ handling can impair cardiac contractile function and increase susceptibility to various cardiovascular diseases (32, 34, 72). The objectives of this study were to establish conditions required to induce regular estrous cycling in grouphoused female mice, to determine whether Ca2⫹ transients and contractions in field-stimulated ventricular myocytes are affected by the estrous cycle, and to determine whether specific cellular mechanisms involved in SR Ca2⫹ release and contraction are modified by the estrous cycle. Studies compared responses in ventricular myocytes and hearts from grouphoused, adult C57BL/6 virgin female mice at all four stages of the estrous cycle. METHODS

Ethical approval. Experiments were performed in accordance with the Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals (CCAC, Ottawa, ON: Vol. 1, 2nd edition, 1993; Vol. 2, 1984). The Dalhousie University Committee on Laboratory Animals approved all experimental protocols used here. C57BL/6 mice were obtained from Charles River Laboratories (St. Constant, QC). Three-month-old virgin female mice were housed in groups of five in micro-isolator cages in the Carleton Animal Care Facility at Dalhousie University on a 12-h light/dark cycle. Food and water were provided to mice ad libitum. Determination of estrous stage. Estrous stages were determined by vaginal cytology with techniques that have been previously described (e.g., 8, 39, 48, 55). Vaginal smears were collected daily between 9:00 am and 11:00 am. Toothpicks and loose cotton were used to create small swabs to obtain vaginal smears. Swabs were dipped in sterile saline before use. Each mouse was held by the base of the tail while the swab was gently inserted into the vaginal opening and rotated to recover cells from the vaginal wall. Cells were then transferred to a slide, allowed to dry, and fixed in 100% methanol. Slides were subsequently stained with eosin and thiazine (Dip Quick stain Set; Jorvet). Stained slides were fixed using a toluene solution (Permount; Fisher Scientific, Ottawa, ON) on a cover slip. In pilot experiments, we found that group-housed female mice did not exhibit regular estrous cycles. Therefore, we induced cycling by exposing mice to bedding collected from the cages of young adult male mice (⬃3 mo). Bedding (⬃50 ml) was placed in cages daily for 7 days before vaginal smear collection, as described previously (10). Both cycling and noncycling mice were swabbed daily for 14 days, and the estrous stages were plotted as a function of time. Day 0 corresponded to the first day that vaginal smears were collected in each group. To determine the estrous stage, vaginal smears were categorized based on the cell types present, as shown in Fig. 1A. Proestrus was identified by the presence of nucleated cells, with or without leukocytes. The estrus stage was characterized by the presence of cornified cells only. Metestrus smears contained mostly lightly stained cornified cells, along with a small number of leukocytes and nucleated cells. Diestrus smears contained leukocytes, along with some nucleated and cornified cells. Although mice occasionally exhibited vaginal smears that were intermediate between two distinct stages (e.g., proestrus/ estrus), these animals were not used in experiments. Figure 1B shows average estradiol levels at each of the estrous stages as reported in previous studies in mice. Values represent estradiol levels in serum

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(22, 77, 82) and plasma (54, 67). This figure shows that the levels of estradiol vary considerably at each estrous stage, although the highest levels occur in proestrus and estrus (Fig. 1B). Figure 1C shows mean levels of serum progesterone at each estrous stage as shown in previous studies in mice (22, 77, 82). The levels of progesterone reported at each stage are less variable between studies, with the highest levels reported in diestrus and the lowest levels in estrus. Ventricular myocyte isolation. Murine ventricular myocytes were isolated as previously described by our group (23). Briefly, mice were anesthetized with pentobarbital sodium (200 mg/kg ip), co-injected with heparin (3,000 U/kg). The heart was removed, the aorta was cannulated, and the heart was perfused for 10 min at 37°C with oxygenated Ca2⫹-free buffer of the following composition (in mM): 105 NaCl, 5 KCl, 1 MgCl2, 0.33 NaH2PO4, 25 HEPES, 20 glucose, 3 Na-pyruvate, and 1 lactic acid (pH 7.4, NaOH). The heart was then perfused for 8 –10 min with Ca2⫹-free buffer supplemented with collagenase type II (8.0 mg/30 ml; Worthington), dispase II (3.4 mg/30 ml; Roche Diagnostics), trypsin (0.5 mg/30 ml; Sigma Aldrich, Oakville, ON), and 50 ␮M CaCl2. The ventricles were cut into small pieces in high-potassium buffer containing (in mM) 45 KCl, 3 MgSO4, 30 KH2PO4, 50 L-glutamic acid, 20 taurine, 0.5 EGTA, 10 HEPES, and 10 glucose (pH to 7.4, KOH). Ventricular tissue was swirled in buffer to dissociate myocytes, and the cell suspension was filtered through a 225 ␮m polyethylene mesh. Only rod shaped myocytes with visible striations were used for experiments. Experimental protocols. Field-stimulation and voltage-clamp experiments were conducted as described in our earlier studies (30, 60). Briefly, ventricular myocytes were loaded with fura-2 AM (5 ␮M) and incubated for 20 min in the dark (20°C). Cells were then placed in a chamber on the stage of an inverted microscope (Nikon Eclipse TE200; Nikon Canada) and superfused (3 ml/min, 37°C) with the following buffer (in mM): 135.5 NaCl, 4 KCl, 1 MgCl2, 10 HEPES, 10 glucose, and 1.0 CaCl2 (pH to 7.4 with NaOH). In most voltageclamp experiments, the buffer solution contained 4-aminopyridine (4 mM) to inhibit transient outward K⫹ current and lidocaine (0.3 mM) to inhibit Na⫹ current. These inhibitors were not present in experiments where cells were voltage-clamped with action potential waveforms. Fluorescence and cell shortening were measured simultaneously by splitting the microscope light between a charge-coupled device camera (Philips, Markham, Ontario) and a photomultiplier tube (Photon Technologies, Birmingham, NJ) with a dichroic cube (Chroma Technology, Rockingham, VT). A DeltaRam fluorescence system (Photon Technologies) alternately excited myocytes at 340 and 380 nm. Fluorescence emitted at 510 nm was recorded for both 340 and 380 nm wavelengths (200 samples/s) with Felix software (PTI). After the experiment, background fluorescence was recorded for each cell and subtracted from fluorescence measurements at each wavelength. These data were expressed as raw fluorescence ratios (F340:F380). Diastolic and systolic Ca2⫹ concentrations were measured, and the difference between these two measures was recorded as the Ca2⫹ transient. Corresponding cell length was measured with a video edge detector (Crescent Electronics, Sandy, UT) at 120 samples/s. Spontaneous activity also was recorded. In field-stimulation experiments, cells were paced with platinum electrodes (3-ms pulses, 4 Hz). Pulses were generated by a stimulus isolation unit (SIU-102; Warner, Hamden, CT) that was controlled with pClamp 8.2 software (Molecular Devices, Sunnyvale, CA). Ten-second recordings were made at 5-min intervals. In current clamp and discontinuous single electrode voltage-clamp (5– 8 kHz) experiments, responses were recorded with microelectrodes (15–25 M⍀; 2.7 M KCl; 2 Hz) and an Axoclamp 2B amplifier operating with pClamp software. In voltage-clamp studies, myocytes were paced with ten 50-ms conditioning pulses from ⫺80 to 0 mV (2 Hz), repolarized to ⫺40 mV, and then depolarized to 0 mV to elicit Ca2⫹ currents, contractions, and Ca2⫹ transients. Action potentials were measured separately, in cells that were not loaded with fura-2. Cells were paced

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00730.2013 • www.ajpheart.org

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with trains of ten 3-ms pulses (2 Hz). SR Ca2⫹ load was measured by rapid application of caffeine (10 mM; 37°C) in cells voltage-clamped as described above. Cells were repolarized to ⫺60 mV and, after 500 ms, caffeine was applied for 1 s in the following buffer (in mM): 10 caffeine, 140 LiCl, 4 KCl, 10 glucose, 5 HEPES, 4 MgCl2, 4 4-aminopyridine, and 0.3 lidocaine. The buffer was Na⫹ and Ca2⫹free to inhibit Ca2⫹ extrusion via Na⫹-Ca2⫹ exchange. Myofilament isolation. Myofilaments were isolated as described previously (85). Mice were anesthetized with pentobarbital sodium as described above, and the ventricles were removed, flash-frozen in liquid nitrogen, and stored at ⫺80°C until use. Ventricles were homogenized in ice-cold buffer as follows (in mM): 60 KCl, 30 imidazole (pH 7.0), 2 MgCl2, 0.01 leupeptin, 0.1 PMSF, 0.2 benzamidine, and 0.1 cantharidin. The homogenate was centrifuged at 14,100 g (15 min, 4°C), and the pellet was resuspended for 45 min in the ice-cold buffer described above plus 1% Triton X-100. This solution was centrifuged at 1,100 g (15 min, 4°C), and the pellet was washed three times in ice-cold buffer. The myofilaments were either stored on ice for a maximum of 2 h or frozen for later use. Actomyosin mg ATPase activity measurement. Actomyosin MgATPase activity was assayed as described previously (85). Myofilaments (25 ␮g) were incubated in solutions containing variable free Ca2⫹ concentrations. Myofilaments were incubated in these solutions for 10 min (32°C), and then reactions were quenched with 10% trichloroacetic acid. The production of inorganic phosphate was measured by adding equal volumes of 0.5% FeSO4 and 0.5% ammonium molybdate in 0.5 M H2SO4. The absorbance was read at 630 nm. Myofilament protein phosphorylation. Cardiac myofilaments (10 ␮g) were resolved using SDS-PAGE (12% resolving) and fixed in 50% methanol-10% acetic acid at room temperature overnight. Total protein phosphorylation was determined by staining gels with ProQ Diamond phosphoprotein stain (Molecular Probes, Eugene, OR). Total protein load was determined by Coomassie staining gels and using actin as a loading control. ProQ Diamond and Coomassie

staining was quantified by imaging gels with a Typhoon gel scanner (GE Healthcare, Baie d=Urfé, PQ), and data were analyzed with ImageJ. Phosphorylation of serines 22 and 23 of cardiac troponin I was assessed by immunoblotting. Myofilament proteins (10 ␮g) were separated by SDS-PAGE (12%) and transferred to nitrocellulose membranes, followed by immunoblotting with an antibody raised against phosphorylated serines 22 and 23 (1:2,000; Cell Signaling, Whitby, ON). Signals were developed using Western Lightning ECL Pro (Perkin Elmer, Woodbridge, ON) and quantified with ImageJ software. Data analysis. Data were analyzed with Clampfit 8.2 (Molecular Devices). Sigma Plot 12.0 (Systat Software) software was used to perform statistical analyses and to create graphs. Myocyte contraction was the difference between resting cell length and cell length at the peak of contraction. Ca2⫹ transients were measured as the difference between diastolic and systolic Ca2⫹ levels. Data are presented as means ⫾ SE. Differences between means were analyzed with a one-way ANOVA. Multiple comparisons were performed using a Holm-Sidak or a Fisher Least Significant Difference post hoc test. Differences in incidence of spontaneous activity were analyzed with a Fisher Exact test. Differences were considered significant if P ⬍ 0.05. Chemicals. Fura-2 AM was obtained from Invitrogen (Burlington, ON) and prepared as a 2-mM stock solution in anhydrous dimethyl sulfoxide. This solution was stored at ⫺20°C until needed. All remaining chemicals were purchased from Sigma-Aldrich. RESULTS

Group-housed female mice do not exhibit regular estrous cycles unless they are induced to cycle. We first investigated whether virgin female mice housed in groups under conventional housing conditions exhibited regular estrous cycles. Vaginal swabs were collected daily for a 2-wk period and stained, as shown in Fig. 1. The estrous stages were plotted as

Fig. 1. Stages of the estrous cycle characterized by vaginal cytology in female mice. A: vaginal smears were stained with eosin and thiazine and classified according to the cell types present on each slide. Slides from mice in proestrus showed mostly nucleated cells with occasional leukocytes, whereas estrus was characterized by densely stained cornified cells. Slides from mice in metestrus contained lightly stained cornified cells plus some leukocytes and nucleated cells. Diestrus was characterized by the presence of leukocytes, as well as some nucleated and cornified cells. B: average levels of estradiol in plasma or serum throughout the estrous cycle in mice (Refs. 22, 54, 67, 77, and 82). C: changes in mean plasma or serum progesterone levels plotted as a function of estrous stage in mice (Refs. 22, 54, 67, 77, and 82). Symbols in B and C correspond to the following studies: Œ ⫽ Ref. 22; e ⫽ Ref. 77; Œ ⫽ Ref. 82;  ⫽ Ref. 67;  ⫽ Ref. 54.

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a function of time (Fig. 2). We found that group-housed female mice did not exhibit regular estrous cycles (Fig. 2A) even though male animals were housed in the same room in close proximity. The noncycling mice spent 19% of their time in proestrus, 13% in estrus, 26% in metestrus, and 53% in diestrus. By contrast, when male bedding was added to their cages to induce cycling, group-housed females consistently exhibited regular 4- to 5-day estrous cycles (Fig. 2B). Thus cycling mice always went through stages in a predictable pattern (proestrus, estrus, metestrus, then diestrus), whereas noncycling mice did not. Similar results were seen in nine noncycling mice and seven cycling mice evaluated for a 2-wk period. These data show that female mice housed in groups do not reliably exhibit regular estrous cycles but that they can be induced to cycle by the addition of male bedding to their cages. The estrous cycle modifies contractions and Ca2⫹ homeostasis in field-stimulated cardiomyocytes. To determine whether the estrous cycle modified contractions and underlying Ca2⫹ transients, responses were recorded simultaneously and compared in field-stimulated myocytes from mice in proestrus, estrus, metestrus, and diestrus. Figure 3A shows representative Ca2⫹ transients (top traces) and contractions (bottom traces) recorded from myocytes isolated during each of the four estrous stages. Responses recorded from the cell isolated from a mouse in estrus were larger than those recorded from cells isolated at any other stage. Figure 3B shows mean (⫾ SE) peak contractions at each estrous stage. Contractions were normalized to resting cell length and expressed as fractional shortening. Fractional shortening was approximately twofold greater in cells from mice in estrus when compared with the other stages (Fig. 3B). Resting cell lengths were not different between groups (data not shown). The velocities of shortening (Fig. 3C) and lengthening (Fig. 3D) were also faster in cells from mice in estrus compared with any other stage. To determine whether an increase in the availability of intracellular Ca2⫹ contributed to the increase in contraction observed in cells from mice in estrus, Ca2⫹ transients were compared at all estrous stages. Figure 4A shows that mean (⫾ SE) Ca2⫹ transients were

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⬃1.11-fold larger in estrus than at any other stage (Fig. 4A). By contrast, resting Ca2⫹ levels were similar, regardless of estrous stage (Fig. 4B). Furthermore, the rates of rise and decay of the Ca2⫹ transient were similar in all four estrous stages (Fig. 4, C and D). The incidence of spontaneous activity was 9.1% in cells isolated from mice in proestrus (n ⫽ 22), 18.5% in estrus (n ⫽ 27), 18.8% in metestrus (n ⫽ 16), and 11.5% in diestrus (n ⫽ 26). Although spontaneous activity occurred most frequently in estrus and metestrus, no significant differences in incidence between groups existed. Together, these observations indicate that peak contractions and underlying Ca2⫹ transients are enhanced in estrus when compared with all other stages of the estrous cycle. Action potentials are prolonged in estrus. The next series of experiments investigated specific cellular mechanisms underlying the increased SR Ca2⫹ release and contraction in estrus. We first compared resting membrane potentials (RMP) and action potential configurations in myocytes isolated from mice at all four estrous stages as described in METHODS. Figure 5A shows representative action potentials recorded from mice in proestrus and estrus. APD was prolonged in the cell from the mouse in estrus when compared with proestrus. Figure 5B shows mean (⫾ SE) values for the RMP in cells from mice in proestrus, estrus, metestrus, and diestrus. RMP was not affected by the estrous stage. Figure 5C shows that mean APD at 50% repolarization (APD50) was longer in estrus when compared with all other stages. APD at 90% repolarization (APD90) also was approximately twofold longer or more in estrus when compared with other estrous stages (Fig. 5D). Because contractions and Ca2⫹ transients recorded from fieldstimulated myocytes are triggered by action potentials, these data demonstrate that the larger responses observed in myocytes isolated from mice in estrus could be due, at least in part, to an increase in Ca2⫹ influx during prolonged depolarization. To test this, a simulated proestrus action potential voltageclamp waveform was created based on mean values for action potentials recorded from myocytes from mice in proestrus, as shown in Fig. 5E. Cells from mice in proestrus and estrus were

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Fig. 2. Mice did not exhibit regular estrous cycles unless they were induced to cycle through exposure to male pheromones. Estrous stages in virgin C57BL/6J were monitored through vaginal cytology for a 2-wk period. A: Three-month-old female mice housed in groups did not exhibit regular estrous cycles. Representative data from 2 mice are shown, but similar results were observed in 9 mice. B: examples of regular estrous cycles observed in group-housed female mice that were exposed to male bedding for 1 wk before collection of vaginal swabs. Similar results were obtained in 7 mice observed over a 2-wk period. P, proesteus; E, estrus; M, metestrus; D, diestrus. In both groups, day 0 corresponds to the first day that vaginal smears were collected.

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Fig. 3. Peak contractions were larger and faster in myocytes from mice in estrus when compared with other stages of the estrous cycle. A: representative examples of Ca2⫹ transients (top) and contractions (bottom) recorded from cells at all 4 estrous stages. Responses were largest in cells from mice in estrus. B: peak contractions, expressed as fractional shortening, were significantly larger in estrus compared with all other stages. Velocities of shortening (C) and lengthening (D) were increased in cells from mice in estrus when compared with other estrous stages. Values represent means ⫾ SE; n ⫽ 27 cells (15 mice) in proestrus, 28 cells (16 mice) in estrus, 26 cells (15 mice) in metestrus, and 25 cells (14 mice) in diestrus. *P ⬍ 0.05.

voltage-clamped with trains of simulated proestrus action potentials. Results showed that differences in peak contractions (Fig. 5F) and Ca2⫹ transients (Fig. 5G) were abolished when estrus and proestrus cells were voltage-clamped with identical action potential waveforms. These findings demonstrate that prolongation of the action potential in estrus contributes to the increase in peak contractions and Ca2⫹ transients observed in this stage. The estrous cycle has no effect on specific EC-coupling mechanisms in voltage-clamped cardiomyocytes. To determine whether the estrous cycle affected components of EC coupling other than APD, the duration of depolarization was controlled by voltage clamp, and responses were compared in myocytes from mice at all four estrous stages. In these experiments, voltage-clamp test steps were preceded by a series of conditioning pulses from ⫺80 to 0 mV to ensure comparable SR Ca2⫹ loading (Fig. 6A, top). Figure 6A shows representative Ca2⫹ transients (top), contractions (middle), and Ca2⫹ currents (bottom) activated by a 200-ms test step from ⫺40 to 0 mV for cells from mice in proestrus and estrus. Mean (⫾ SE) data demonstrate that peak Ca2⫹ transients, Ca2⫹ currents, and

contractions were similar at all four stages of the estrous cycle (Fig. 6, B-D). Furthermore, the gain of EC coupling, calculated as amount of Ca2⫹ released per unit Ca2⫹ current, was not affected by the estrous stage (Fig. 6E). The resting Ca2⫹ levels in diastole were also similar in proestrus, estrus, metestrus, and diestrus (Fig. 6F). To determine whether the amount of SR Ca2⫹ available for release was modified by the estrous cycle, SR Ca2⫹ content was measured by the rapid application of caffeine as described in METHODS. Figure 7A shows representative Ca2⫹ transients induced by caffeine in myocytes from mice in proestrus and estrus. The mean (⫾ SE) data demonstrate that SR Ca2⫹ content was similar at all stages of the estrous cycle (Fig. 7B). Fractional release, which was calculated as Ca2⫹ release/the total amount of SR Ca2⫹ available, was also similar in all four groups (Fig. 7C). These data show that the increase in SR Ca2⫹ release and contraction in estrus was not due to changes in peak Ca2⫹ current, the gain of SR Ca2⫹ release, or the amount of SR Ca2⫹ available for release. Myofilament Ca2⫹ sensitivity is lowest in estrus. The estrous cycle could potentially influence contractile function at the level of the cardiac myofilaments. To determine whether myo-

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00730.2013 • www.ajpheart.org

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Fig. 4. Ca transients were increased in estrus when compared with the other 4 stages of the estrous cycle. A: peak Ca2⫹ transients were significantly increased in estrus when compared with other stages. B: diastolic Ca2⫹ levels did not vary throughout the estrous cycle. The rate of rise (C) and the decay rate (D) were not affected by the estrous cycle. Values represent means ⫾ SE; n ⫽ 25 cells (14 mice) in proestrus, 27 cells (14 mice) in estrus, 17 cells (11 mice) in metestrus, and 26 cells (15 mice) in diestrus. *P ⬍ 0.05.

filament Ca2⫹ sensitivity was affected by the estrous cycle in cardiomyocytes, we plotted contractions recorded from fieldstimulated cells as a function of the cytosolic Ca2⫹ concentration to create phase-loop plots. Previous studies have shown that relaxation phase of the loop provides an estimate of the responsiveness of myofilaments to intracellular Ca2⫹ (73). Representative phase-loop plots were shifted to the right in the examples from mice in estrus, proestrus, and diestrus when compared with metestrus (Fig. 8A), which suggests that cells from mice in proestrus, estrus, and diestrus had lower myofilament Ca2⫹ sensitivity than the cell in metestrus. This finding was quantified by measuring the cytosolic Ca2⫹ concentration at 50% cellular relaxation (data in Figs. 3B and 4A) to provide an estimate of myofilament Ca2⫹ sensitivity, as in previous studies (51, 60). The mean (⫾ SE) data show that the Ca2⫹ concentration at 50% relaxation was significantly higher in myocytes from mice in estrus and proestrus when compared with metestrus (Fig. 8B). These data suggested that myofilament Ca2⫹ sensitivity might fluctuate with the estrous cycle. To directly measure myofilament Ca2⫹ sensitivity, actomyosin MgATPase activity was measured in the ventricles as

described in METHODS. Maximal actomyosin MgATPase activity and the Hill coefficients were similar in all four groups (Table 1), so data were normalized to maximum to facilitate comparisons of myofilament Ca2⫹ sensitivity between groups. Figure 8C shows the average (⫾ SE) normalized actomyosin MgATPase activity plotted as a function the Ca2⫹ concentration for hearts at each of the four estrous stages. The curve for the hearts in estrus was shifted to the right (inset, Fig. 8C), which indicates a reduction in myofilament sensitivity to Ca2⫹. This was quantified by comparing the concentration of Ca2⫹ required to produce a 50% increase in activity (EC50 values) between groups as shown in Fig. 8D. We found that the EC50 values were significantly larger in estrus when compared with other stages (Fig. 8D). These results demonstrate that myofilament Ca2⫹ sensitivity was lowest in estrus. Taken together, our results show that although myofilaments from mice in estrus were the least sensitive to Ca2⫹, contractions remained larger than in other stages. Myofilament protein phosphorylation. Cardiac myofilament function is profoundly influenced by phosphorylation changes in key myofilament proteins. Therefore, phosphorylation levels

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H944

CONTRACTION FLUCTUATES WITH THE ESTROUS CYCLE

A

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Proestrus

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Estrous Stage

Proestrus

Estrus

Estrous Stage

Fig. 5. The action potential was prolonged in estrus, when compared with all other stages of the estrous cycle. A: representative examples of action potentials recorded from ventricular myocytes isolated from mice at all 4 stages of the estrous cycle. The action potential duration (APD) prolonged in estrus when compared with the other stages. B: resting membrane potential (RMP) was similar regardless of estrous stage. C: mean ⫾ SE values for APD at 50% repolarization (APD50) were longer in estrus than in the other estrous stages. D: APD at 90% repolarization (APD90) also was significantly prolonged in cells from mice in estrus when compared with all other estrous stages (n ⫽ 6 cells in proestrus, 7 in estrus, 7 in metestrus, and 4 in diestrus). E: simulated proestrus action potential based on the average values for action potential configuration recorded in myocytes from mice in proestrus. F and G: peak contractions and Ca2⫹ transients were similar in myocytes from mice in proestrus and estrus when cells were voltage-clamped with a simulated proestrus action potential (n ⫽ 4 cells in proestrus and 9 in estrus for Ca2⫹ transients and n ⫽ 4 cells in proestrus and 8 in estrus for contractions). Values represent means ⫾ SE; cells were obtained from 2 mice/group. *P ⬍ 0.05.

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H945

CONTRACTION FLUCTUATES WITH THE ESTROUS CYCLE 0

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-6

Estrous stage Fig. 6. Mechanisms of excitation-contraction (EC) coupling were not influenced by the estrous cycle. A: voltage-clamp protocol is shown at top. Ca2⫹ transients, contractions, and Ca2⫹ currents were activated simultaneously by a test step to 0 mV; test steps were preceded by 10 conditioning pulses from ⫺80 to 0 mV. Representative Ca2⫹ transients (top), contractions (middle), and Ca2⫹ currents (bottom) recorded from myocytes at all estrous stages are shown. B: when cells were voltage-clamped to control the duration of depolarization, the magnitude of Ca2⫹ transients was not affected by the estrous cycle. Peak Ca2⫹ currents (C) and contractions (D) in voltage-clamped cells were unaffected by the estrous cycle. E: gain of EC coupling (Ca2⫹ release/unit Ca2⫹ current) was similar at all 4 stages of the estrous cycle. F: diastolic Ca2⫹ levels did not vary with the estrous stage. Values represent means ⫾ SE; n ⫽ 13 cells (6 mice) in proestrus, 12 cells (5 mice) in estrus, 13 cells (7 mice) in metestrus, and 13 cells (6 mice) in diestrus.

of critical myofilament proteins were compared at all four estrous stages. Results showed that myosin binding protein C phosphorylation was highest during estrus (Fig. 9, A and B), which corresponded to the stage with the lowest myofilament

Ca2⫹ sensitivity and the highest myocyte contractility. Furthermore, this increase in phosphorylation of myosin binding protein C in estrus was significant when compared with metestrus (Fig. 9B). By contrast, phosphorylation levels for other

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H946

CONTRACTION FLUCTUATES WITH THE ESTROUS CYCLE

A

0 -40 mV -80 1s Caffeine

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Estrous stage 2⫹

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Estrus

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Estrous stage 2⫹

Fig. 7. Sarcoplasmic reticulum (SR) Ca content and fractional Ca release did not vary with the estrous cycle. The voltage-clamp protocol is shown at top. SR Ca2⫹ content was assessed by the rapid application of 10 mM caffeine; caffeine application was preceded by 10 conditioning pulses from ⫺80 to 0 mV. A: representative caffeine-induced Ca2⫹ transients recorded from myocytes in proestrus and estrus preceded by responses activated by conditioning pulses. Arrow indicates caffeine-induced Ca2⫹ transients. B: SR Ca2⫹ content was similar at all 4 estrous stages. C: fractional release of SR Ca2⫹ (Ca2⫹ release/total SR Ca2⫹ available for release) did not vary with the estrous stage. Values represent means ⫾ SE; n ⫽ 4 cells (3 mice) in proestrus, 6 cells in estrus (3 mice), 7 cells (5 mice) in metestrus, and 9 cells (5 mice) in diestrus.

myofilament proteins (desmin, troponin T, tropomyosin, and troponin I) were similar in all four groups (Fig. 9, A and B). Phosphorylation of the NH2-terminal serines in troponin I reduces myofilament Ca2⫹ sensitivity but enhances myocyte contractility, as observed in hearts from mice in estrus in our study. Therefore, immunoblots for changes in the phosphorylation of these residues also were compared at all four stages. Figure 9C shows that although the highest levels of phosphorylation occurred in proestrus and estrus, these differences were not statistically significant. DISCUSSION

The overall goals of this study were to establish conditions required to induce regular estrous cycling in group-housed female mice and to evaluate the impact of the estrous cycle on contractions, Ca2⫹ transients, and underlying EC-coupling mechanisms. Results showed that female mice did not exhibit regular estrous cycles unless they were induced to cycle by the addition of male bedding to their cages. When mice were induced to cycle, components of EC coupling were modified in

ventricular myocytes from animals in estrus compared with the other stages of the estrous cycle. Field-stimulated myocytes from mice in estrus had larger contractions and Ca2⫹ transients and cells exhibited prolonged action potentials. Interestingly, the impact of estrus on peak contractions and Ca2⫹ transients was eliminated in voltage-clamp studies when cells were depolarized with identical action potential waveforms. Voltageclamp experiments also revealed that the stage of the estrous cycle had no effect on cellular mechanisms that regulate SR Ca2⫹ release, including Ca2⫹ current, EC-coupling gain, diastolic Ca2⫹, SR Ca2⫹ content, or fractional SR Ca2⫹ release. Even though contractions were largest in estrus, myofilament Ca2⫹ sensitivity was lower in estrus than in other stages in conjunction with higher levels of myosin binding protein C phosphorylation. Contractions were enhanced by an increase in APD and subsequent increase in SR Ca2⫹ release. These results show that cyclical changes in reproductive hormones associated with the estrous cycle can influence myocardial electrical and contractile function and modify Ca2⫹ homeostasis. However, such changes are unlikely to occur in female

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H947

CONTRACTION FLUCTUATES WITH THE ESTROUS CYCLE

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Fig. 8. Myofilament Ca sensitivity was lower in estrus than at any other stage of the estrous cycle. A: representative contraction-[Ca2⫹] phase loop plots for cells at all 4 estrous stages. In each case, the curve represents the average of ⬃30 responses from an individual cardiomyocyte at each stage. A rightward shift in the curve indicates a decrease in myofilament Ca2⫹ sensitivity. B: Ca2⫹ levels at 50% relaxation, used to quantify this shift, were significantly higher in proestrus and estrus when compared with metestrus. C: myofilament Ca2⫹ sensitivity was measured directly with an actomyosin MgATPase activity assay. Responses were normalized to maximal actomyosin MgATPase activity in each group. Myofilament Ca2⫹ sensitivity was lowest in estrus, as indicated by a rightward shift in the actomyosin MgATPase activity-[Ca2⫹] curve (detail shown in inset; *significant difference between estrus and metestrus; †significant difference between estrus and proestrus). D: EC50 values for Ca2⫹ were significantly higher in estrus when compared with all other stages. Values represent means ⫾ SE. In B, n ⫽ 11 cells (8 mice) in proestrus, 12 cells (9 mice) in estrus, 10 cells (5 mice) in metestrus, and 13 cells (10 mice) in diestrus; for C and D, n ⫽ 7 hearts from 7 mice in each group. *P ⬍ 0.05.

mice housed in groups under conventional conditions since these mice do not exhibit regular cycling. Despite growing evidence that physiology and disease expression differs between the sexes, considerable bias against Table 1. Actomyosin MgATPase activity at various stages of the estrous cycle Estrous Stage

Maximal Actomyosin MgATPase Activity, nM Pi·min⫺1·mg protein⫺1

Hill Coefficient

Proestrus Estrus Metestrus Diestrus

193.8 ⫾ 4.2 197.6 ⫾ 4.9 202.3 ⫾ 3.1 192.4 ⫾ 4.7

1.90 ⫾ 0.08 2.03 ⫾ 0.04 2.05 ⫾ 0.05 2.06 ⫾ 0.06

Values are means ⫾ SE; for all stages, n ⫽ 7 hearts.

the use of female animals in all fields of biomedical research including the cardiovascular field exists (3, 69, 76, 88). One reason for this bias is concern about the confounding effect of cyclical changes in female reproductive hormones (3, 69, 88). A key observation in our study is that virgin female mice housed in groups under conventional animal care conditions did not exhibit regular estrous cycles. This observation is likely due to the inhibitory effect of female pheromones on the estrous cycle, a phenomenon known as the Lee-Boot effect, which was first described in the 1950s but is not often discussed in current literature (40, 41, 81). Suppression of the estrous cycle in grouphoused female mice may explain why many studies have concluded that results obtained from females are not inherently more variable than results from studies that used males (reviewed by 3, 88). Thus concerns about potential effects of the estrous cycle on

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H948

CONTRACTION FLUCTUATES WITH THE ESTROUS CYCLE

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experimental results obtained in female mice may be unfounded, unless the animals are specifically induced to cycle by exposure to male pheromones (36, 80). A novel observation reported here is that when mice were cycling regularly, contractions and associated Ca2⫹ transients were larger in field-stimulated ventricular myocytes from mice in estrus than in cells isolated from mice at any other estrous stage. Because contraction is proportional to the magnitude of

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Fig. 9. Changes in myofilament protein phosphorylation across estrous stages. A: myofilament proteins separated by SDS-PAGE were stained with ProQ Diamond (left) to assess myofilament protein phosphorylation. Total protein load was determined by subsequently staining gels with coomassie (middle). Protein marker (right) shows approximate molecular weight of myofilament proteins. B: quantification of myofilament protein phosphorylation changes revealed a significant increase in total myosin binding protein C phosphorylation during estrus as compared with metestrus. There were no other statistically significant differences in phosphorylation status between the various estrous stages. C: phosphorylation of the NH2-terminal serines 22/23 in troponin I was determined with an immunoblot using a phosphor-specific antibody (left) and normalized to total troponin I levels. Quantification (right) showed no significant differences in NH2-terminal TnI phosphorylation across estrous stages, although levels were higher in proestrus and estrus when compared with the other stages. MyBP-C, myosin binding protein C; TnT, troponin T; TnI, troponin I. Values represent means ⫾ SE; n ⫽ 5 hearts from 5 mice in each group. *P ⬍ 0.05. AU, arbitrary units.

E

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the Ca2⫹ current that triggers SR Ca2⫹ release (5), an increase in Ca2⫹ current could, in theory, account for the increase in SR Ca2⫹ release observed in estrus. Indeed, a previous report showed that Ca2⫹ current was larger in estrus compared with diestrus in ventricular myocytes isolated from young (6 wk old) female guinea pigs (31). By contrast, we found that the magnitude of the Ca2⫹ current was similar at all four stages of the estrous cycle in myocytes from 3-mo-old virgin female

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CONTRACTION FLUCTUATES WITH THE ESTROUS CYCLE

mice. Although it is possible that the impact of the estrous cycle on transmembrane currents differs between species, it also may depend on age or whether virgin animals were used. In theory, larger Ca2⫹ transients in estrus could be explained by increased EC-coupling gain, higher SR Ca2⫹ content, increased diastolic Ca2⫹, and/or an increase in the fractional release of SR Ca2⫹. However, our study demonstrates that these critical EC-coupling mechanisms do not fluctuate with the estrous cycle; therefore other underlying mechanisms were investigated. It is possible that larger Ca2⫹ transients in estrus arose from prolongation of the APD. Longer action potentials increase the duration of depolarization and enhance Ca2⫹ influx (12). This would be expected to augment SR Ca2⫹ release and increase contractions in field-stimulated myocytes, where responses are activated by action potentials. Interestingly, we found that APD was markedly prolonged in ventricular myocytes from mice in estrus when compared with diestrus, as shown previously in cardiomyocytes from mice and guinea pigs (31, 67). However, our study extends previous observations to demonstrate that APD was prolonged in estrus when compared with all other stages of the estrous cycle. The increase in APD in estrus has been attributed to a reduction in fast-transient outward K⫹ current (Ito,f) as well as a decrease in the ultrarapid delayed rectifier K⫹ current (IK,slow) in mice (67), but an increase in Ca2⫹ current density in guinea pigs (31). Our study demonstrates that an increase in Ca2⫹ current density does not contribute to prolongation of the action potential in cells from mice in estrus. Furthermore, when cells were voltage-clamped with simulated proestrus waveforms, peak contractions, and Ca2⫹ transients were similar in cells from mice in proestrus and estrus. These data demonstrate that prolongation of the APD in estrus enhances SR Ca2⫹ release and augments contractions in isolated cardiomyocytes. Previous studies have shown that the levels of estradiol rise in proestrus and peak before ovulation in the mouse model (22, 77, 82). Therefore, myocytes have been exposed to the highest levels of circulating estradiol for the longest time in late proestrus and estrus. One might speculate that the increase in contractions, Ca2⫹ transients, and APD arises as a result of prolonged exposure to high levels of circulating estradiol. Some support for this in the literature exists. For example, transcript levels of Kv4.3 and Kv1.5 (underlying Ito,f and IK,slow) decline and APD is prolonged in ovariectomized (OVX) mice treated with high levels of estradiol (67). This suggests that prolonged exposure to high levels of estradiol can recapitulate the changes in APD observed in mice in estrus. Our observation that the largest Ca2⫹ transients are seen in estrus, following prolonged estradiol exposure, contrasts with the results of most previous studies in OVX animals. Studies have shown that OVX, which dramatically reduces circulating estradiol levels, increases contractions and Ca2⫹ transients in rodent models (13, 19, 37, 46, 83) (c.f. 7, 64). Together with our present data, these findings suggest that the influence of reproductive hormones on cardiomyocyte EC coupling is complex. Indeed, the estrus stage is characterized by elevated levels of follicle-stimulating hormone and luteinising hormone, as well as very low levels of progesterone (22, 59, 77, 82). Thus, the progesterone-to-estradiol ratio, rather than the absolute levels of these hormones, may be responsible for the influence of sex hormones on the heart (31, 67). Alternatively, follicle-

H949

stimulating hormone, luteinising hormone, and/or progesterone may affect myocardial function, although this has not yet been investigated. Whether circulating levels of estradiol peak in proestrus or in estrus in the mouse model is not clear. Although some studies report peak levels in estrus (22, 82), others indicate that the highest levels occur in proestrus (54, 67, 77). Furthermore, as shown in Fig. 1B, the circulating levels of estradiol at each stage vary highly between studies. Several factors likely contribute to this variability. First, previous studies used different experimental approaches to measure circulating estradiol levels, so results may not be directly comparable. For example, Nelson et al. (54) employed radioimmunoassays, whereas other studies used enzyme immunoassays (22, 82) or fluoroimmunoassays (77). Furthermore, because circulating estradiol levels are very low in the mouse model, studies suggest that it is difficult to accurately and consistently measure estradiol in mouse blood samples (26, 49). In fact, whether estradiol can be reliably measured in blood samples from mice with any experimental approach is controversial (26, 49). The results of our in vitro experiments clearly demonstrate that myofilament Ca2⫹ sensitivity was influenced by the estrous cycle. Interestingly, myofilament Ca2⫹ sensitivity was lowest in estrus when compared with all other stages, even though contractions were actually largest at this stage. This reduction in myofilament Ca2⫹ sensitivity in estrus may be linked to prolonged exposure to high levels of circulating estradiol. Indeed, acute activation of estrogen receptor alpha has been shown to reduce myofilament Ca2⫹ sensitivity (38). Furthermore, evidence that myofilament Ca2⫹ sensitivity increases when estradiol levels are reduced following OVX (7, 78) exists, although this has not been observed in all studies (62, 68). Thus it is possible that exposure to increasing levels of estradiol during the follicular phase of the estrous cycle reduces myofilament Ca2⫹ sensitivity. In contrast with our in vitro findings, the evidence for a reduction in myofilament Ca2⫹ sensitivity in cardiomyocytes from mice in estrus was less convincing. Analysis of phaseloop plots suggested that myocytes from mice in estrus and proestrus had the lowest Ca2⫹ sensitivity, and this was significant only compared with metestrus. Several reasons could account for differences in results between our in vitro studies and our cellular studies. First, it is important to note that phase-loop plots provide only an estimate of myofilament Ca2⫹ sensitivity, and it may be that myofilament Ca2⫹ sensitivity is reduced in estrus in vitro but not in isolated myocytes. Indeed, isolated cardiomyocytes differ from the in vivo heart in important ways that may affect their behavior. For example, isolated cardiomyocytes are enzymatically digested and separated from neighboring cells. Furthermore, although they are a widely used and accepted model for cardiac function (16), they are not subject to stretch and do not work against a load. Additional experiments that explore the effect of the estrous cycle on cardiac contractile function in vivo and on cardiomyocytes subjected to stretch could be revealing. Our data demonstrate both an increase in myocyte contractility and a decrease in myofilament Ca2⫹ sensitivity during the estrus phase. These observations may seem contradictory, since reduced myofilament Ca2⫹ sensitivity should attenuate force development (5). However, previous studies have reported similar results. For example, it is well known that

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H950

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␤-adrenergic stimulation of the heart activates PKA, which increases peak force but reduces myofilament Ca2⫹ sensitivity through phosphorylation of the NH2-terminal serines in troponin I (87). Subsequent studies also have linked increased myosin binding protein C phosphorylation with lower myofilament Ca2⫹ sensitivity, along with accelerated stretch activation and increased systolic force (74). The relative contributions of these changes are not clear, although some have suggested a collaborative relationship (75). We report a significant increase in myosin binding protein C phosphorylation in conjunction with a small increase in NH2-terminal troponin I phosphorylation in hearts isolated from mice in estrus. These changes were associated with reduced myofilament Ca2⫹ sensitivity, enhanced fractional shortening, and increased contraction velocities. Because the increase in NH2-terminal troponin I phosphorylation in estrus was not statistically significant, our results suggest that myosin binding protein C phosphorylation may be sufficient to reduce myofilament Ca2⫹ sensitivity. These observations agree with the results of Chen et al. (11) who reported similar findings with respect to myosin binding protein C phosphorylation and the results of Stelzer et al. (74) who showed that the associated increase in myocyte contractile function is the result of accelerated stretch activation. This reduction in myofilament Ca2⫹ sensitivity in estrus may counteract the larger Ca2⫹ transients and preserve cardiac contractility in vivo, although additional studies will be required to investigate this further. Variations in the levels of reproductive hormones during the menstrual cycle can affect susceptibility to cardiovascular disease in premenopausal women. Ischemic episodes occur more frequently and are more severe during the luteal phase of the menstrual cycle, when progesterone levels are high (33). In addition, ischemic injury and myocardial infarction are most easily induced in the early follicular phase, when estradiol levels are low (43, 52). Cyclical changes in the levels of reproductive hormones can also modify susceptibility to cardiovascular diseases in rodent models. For example, hearts from rats in proestrus are resistant to the adverse effects of hypoxia and trauma-hemorrhage (6, 84). Taken together, these observations suggest that susceptibility to cardiovascular disease may rise when progesterone levels are high and estradiol levels are low. Intracellular Ca2⫹ overload at the level of the cardiomyocyte is known to play an important role in the pathogenesis of many different cardiovascular diseases (72, 79). However, we found that the highest levels of intracellular Ca2⫹ occurred in estrus, when cells had been exposed to high levels of estradiol for the longest time. Thus our data suggest that elevated intracellular Ca2⫹ levels in cardiomyocytes do not account for variations in the expression of cardiovascular disease associated with the estrous or menstrual cycles. Still, our observation that cells from mice in estrus have double the likelihood of spontaneous activity compared with proestrus is compatible with a previous report that hearts from rats in proestrus are slow to develop ventricular fibrillation following coronary ligation compared with other stages (27). Interestingly, earlier studies in mice have shown that myofilament Ca2⫹ sensitivity can influence susceptibility to cardiac arrhythmias. Specifically, agents that increase myofilament Ca2⫹ sensitivity can promote cardiac arrhythmias, whereas agents that desensitize the myofilaments prevent the induction of arrhythmias (2). Thus the reduction in

myofilament Ca2⫹ sensitivity that we observed in hearts from mice in estrus may contribute to cardioprotective effects associated with high estradiol levels. In the present study, we expressed our Ca2⫹ transient data as raw fluorescence ratios, rather than Ca2⫹ concentrations. This approach is frequently used in studies of Ca2⫹ handling in cardiac myocytes (e.g., 18, 42, 58) due to limitations associated with in vitro and in vivo calibration of fura-2 AM. In vitro calibration does not account for binding of the dye to proteins (35), so it may underestimate intracellular Ca2⫹, as shown experimentally in previous studies (4). On the other hand, in vivo calibration may overestimate intracellular Ca2⫹ due to factors such as incomplete hydrolysis leading to Ca2⫹-insensitive fluorescence (28) and loading of the dye into organelles (45). If we use an in vitro calibration curve to convert our data to Ca2⫹ concentration, peak Ca2⫹ transients are ⬃70 nM in proestrus, metestrus, and diestrus compared with ⬃90 nM in estrus. These values are comparable with those previously reported by our group and others who have used in vitro calibration curves to investigate Ca2⫹ handling in myocytes from female rodents (e.g., 14, 20, 66). By contrast, with a similar calibration approach, peak transients are much larger in myocytes from male rodents (e.g., 15, 50, 56, 57). Clearly, future studies of Ca2⫹ handling in cardiomyocytes must indicate the sex of the animals used and data from male and female animals should not be pooled. There are limitations to the approaches used in this study. Our study investigated the impact of the estrous cycle on ventricular myocytes, so whether the estrous cycle can affect contractions and Ca2⫹ homoestasis in atrial myocytes is not yet known. Our experiments investigated cells at physiological temperature (37°C) and rapid pacing rates (2– 4 Hz). The persistent rapid pacing of cells in field-stimulation experiments resulted in relatively high levels of diastolic Ca2⫹ in our experiments, as shown by others previously (17). The peak Ca2⫹ transients and contractions reported in our study were relatively small, as in previous studies that used similar temperature and pacing conditions (e.g., 19, 21, 30, 60). By contrast, much larger Ca2⫹ transients and contractions are recorded in cardiomyocytes investigated at room temperature, which is commonly used in studies of EC coupling (63, 70, 71). We also found that contractions measured in field stimulation experiments were smaller than those recorded under voltage-clamp conditions. This likely reflects differences in the activating stimulus, as cells were activated by brief action potentials in field-stimulation studies in comparison with the 200-ms test step used in our voltage-clamp experiments. In summary, the present study demonstrated that the estrous cycle modified the amplitudes of contractions and Ca2⫹ transients in isolated mouse ventricular myocytes. Peak responses were larger in estrus than at any other stage, and this increase was mediated, in part, by an increase in APD. Interestingly, although contractions were large, myofilament Ca2⫹ sensitivity was lowest in estrus and contractions were enhanced by increased intracellular Ca2⫹. By contrast, the estrous cycle had no effect on other key mechanisms that regulate cardiac EC coupling, such as Ca2⫹ current, EC-coupling gain, or SR Ca2⫹ stores. This study also demonstrated that female mice did not exhibit regular estrous cycles unless they were specifically induced to do so. Furthermore, the only stage where changes in EC coupling occurred was estrus. Still, mice spend less than

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10% of their total reproductive cycle in estrus (1, 29) and we found that noncycling mice exhibited vaginal cytology characteristic of estrus infrequently. Thus the chance that a mouse used in any given experiment will be in the estrus stage is very low. Together, these observations indicate that although the estrous cycle can influence cardiac EC-coupling, concerns about the influence of the estrous cycle on results from female animals may be unwarranted. ACKNOWLEDGMENTS We thank Dr. Jie-quan Zhu and Peter Nicholl for excellent technical assistance, Dr. Robert Rose for valuable comments on an earlier version of this manuscript, and Randi J Parks for helpful comments on the data presented in this manuscript. GRANTS This study was supported by grants from the Canadian Institutes for Health Research (MOP 97973; to S. E. Howlett) and Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (RGPIN 261928; to W. G. Pyle). J. K. MacDonald received studentship support from the Nova Scotia Health Research Foundation. C. J. Reitz was supported with an NSERC undergraduate research assistantship. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: J.K.M. and S.E.H. conception and design of research; J.K.M., W.G.P., C.J.R., and S.E.H. performed experiments; J.K.M., W.G.P., C.J.R., and S.E.H. analyzed data; J.K.M., W.G.P., C.J.R., and S.E.H. interpreted results of experiments; J.K.M., W.G.P., C.J.R., and S.E.H. prepared figures; J.K.M. and S.E.H. drafted manuscript; J.K.M., W.G.P., C.J.R., and S.E.H. edited and revised manuscript; J.K.M., W.G.P., C.J.R., and S.E.H. approved final version of manuscript. REFERENCES 1. Barkley MS, Bradford GE. Estrous cycle dynamics in different strains of mice. Proc Soc Exp Biol Med 167: 70 –77, 1981. 2. Baudenbacher F, Schober T, Pinto JR, Sidorov VY, Hilliard F, Solaro RJ, Potter JD, Knollmann BC. Myofilament Ca2⫹ sensitization causes susceptibility to cardiac arrhythmia in mice. J Clin Invest 118: 3893–3903, 2008. 3. Beery AK, Zucker I. Sex bias in neuroscience and biomedical research. Neurosci Biobehav Rev 35: 565–572, 2011. 4. Berlin JR, Konishi M. Ca2⫹ transients in cardiac myocytes measured with high and low affinity Ca2⫹ indicators. Biophys J 65: 1632–1647, 1993. 5. Bers DM. Calcium cycling and signaling in cardiac myocytes. Annu Rev Physiol 70: 23–49, 2008. 6. Broderick TL, Wong P. Influence of the estrous cycle on hypoxic failure in the female rat heart. Gend Med 6: 596 –603, 2009. 7. Bupha-Intr T, Wattanapermpool J, Peña JR, Wolska BM, Solaro RJ. Myofilament response to Ca2⫹ and Na⫹/H⫹ exchanger activity in sex hormone-related protection of cardiac myocytes from deactivation in hypercapnic acidosis. Am J Physiol Regul Integr Comp Physiol 292: R837–R843, 2007. 8. Byers SL, Wiles MV, Dunn SL, Taft RA. Mouse estrous cycle identification tool and images. PLoS One 7: e35538, 2012. 9. Caligioni CS. Assessing reproductive status/stages in mice. Curr Protoc Neurosci Appendix 4: Appendix 4I, 2009. 10. Campbell CS, Ryan KD, Schwartz NB. Estrous cycles in the mouse: relative influence of continuous light and the presence of a male. Biol Reprod 14: 292–299, 1976. 11. Chen PP, Patel JR, Rybakova IN, Walker JW, Moss RL. Protein kinase A-induced myofilament desensitization to Ca2⫹ as a result of phosphorylation of cardiac myosin-binding protein C. J Gen Physiol 136: 615–627, 2010. 12. Clark RB, Bouchard RA, Giles WR. Action potential duration modulates calcium influx, Na⫹-Ca2⫹ exchange, and intracellular calcium release in rat ventricular myocytes. Ann N Y Acad Sci 779: 417–429, 1996.

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