Heart and Circulatory Physiology

Mechanisms of sex differences in rat cardiac myocyte response to β-adrenergic stimulation

Vida M. Vizgirda, Gordon M. Wahler, Korie L. Sondgeroth, Mark T. Ziolo, Dorie W. Schwertz


The purpose of this study was to investigate sex differences in the functional response of isolated rat heart ventricular myocytes to β-adrenergic stimulation and in isoproterenol-stimulated signal transduction. Fractional shortening was measured using a video edge-detection system in control- and isoproterenol-stimulated myocytes that had been isolated from weight-matched rats. Number and affinity of the β-adrenergic receptors and the L-type Ca2+ channel were measured in ventricular cardiac membranes by radioligand binding studies. Control- and isoproterenol-mediated alteration in Ca2+ current density (I Ca) was determined by patch clamping and cellular cAMP content was determined by radioimmunoassay. Study results demonstrate that female myocytes have higher Ca2+channel density and greater I Ca than male myocytes. However, isoproterenol elicits a greater β-adrenergic receptor-mediated increase cell shortening, I Caand cAMP production in male myocytes. Male myocytes were also found to have a higher β-adrenergic receptor density. These results suggest that cardiac myocytes from male rats have an enhanced response to β-adrenergic stimulation due to augmented β-adrenergic signaling that results in a greater transsarcolemmal Ca2+ influx.

  • gender
  • calcium channel
  • calcium current
  • isoproterenol
  • heart

the results of many investigations document gender differences in basal cardiac function (14, 15, 29, 30), response to increased physiological load (exercise) (1, 7, 8, 16, 17, 29) and pathological pressure overload (6, 24, 43). The sympathetic nervous system plays a central role in regulating heart function and response to load stress through β-adrenergic receptor stimulation. Binding of β-adrenergic agonists to receptors in the heart activates adenylyl cyclase via a stimulatory G protein. The resultant production of the second messenger cAMP activates protein kinase A and the kinase phosphorylates cellular proteins including the α1c subunit of the L-type Ca2+ channel (10, 19). The result is an increase in the inward Ca2+ current (I Ca) and increased force of contraction.

Animal studies (4, 27, 34, 35, 36) confirm gender differences in myocardial function and response to exercise and pathophysiological load and provide models for investigation of mechanisms responsible for these sex differences. Using an isolated atrial model, our laboratory (37) recently described sex differences in contractile function and in response to β-adrenergic stimulation. The primary purpose of this study is to investigate sex differences in isolated rat ventricular myocyte response to β-adrenergic stimulation. Our study is the first to use isolated ventricular cells to study sex differences in response to β-adrenergic stimulation in the absence of neurohormonal and hemodynamic influences. Specifically, we investigated potential sex differences in 1) control (field stimulated in the absence of drug) and isoproterenol-stimulated ventricular myocyte shortening,2) β-adrenergic receptor and L-type Ca2+channel density and affinity, 3) control and isoproterenol-stimulated myocyte cAMP content, and 4) control and isoproterenol-stimulated I Ca. Theoretically, sex differences in response to β-adrenergic agonists could contribute to gender differences in heart function and pathology.



Sexually mature, weight-matched (270–290 g weight range) male and female Sprague-Dawley rats were used in all the studies. Same-sex animals were housed three to a cage with free access to water and rat chow and were maintained on 12-h day/night cycles. Treatment of animals conformed to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1985).

Myocyte isolation methods.

Ventricular myocytes for shortening studies were isolated by an enzymatic dissociation procedure as previously described by Pyo and Wahler (31). Thirty minutes before the heart was removed, animals were anesthetized with pentobarbital sodium (50 mg/kg ip) and heparinized (200 U ip) to prevent coagulation. The heart was removed via sternotomy and placed in cold (4°C) nominally Ca2+-free solution containing (in mM) 133.5 NaCl, 1.2 NaH2PO4, 4.0 KCl, 1.2 MgSO4, 10.0 HEPES, 11.1 glucose, and 1 mg/ml fatty acid-free albumin (pH 7.4). The heart was retrogradely perfused for 5 min with oxygenated (100% O2) nominally Ca2+-free solution (37°C), followed by perfusion (16.5 min/g wet wt) with the same solution containing 5 μM CaCl2 and 50 U/ml of type II collagenase (pH 7.4) (Worthington Biochemical; Freehold, NJ). Ventricular tissue was then removed and periodically triturated for 10 min in warm (37°C) storage solution (Ca2+-free solution + 50 μM CaCl2) to obtain single myocytes. Isolated myocytes were resuspended in centrifuge tubes and allowed to settle for 15 min, and the supernatant was then discarded. This procedure was repeated once more, and the final cell pellet was resuspended in storage solution.

Ventricular myocytes for electrophysiologic studies were isolated in a similar method with the following modifications (20). Isolated hearts were briefly perfused with a modified warmed, oxygenated (37°C, 95% O2-5%CO2) nominally Ca2+-free buffer containing (in mM) 118.5 NaCl, 14.5 NaHCO3, 2.6 KCl, 1.18 MgCl2, 11.1 glucose, and 1 mg/ml fatty acid free albumin (pH 7.4), followed by a 16.5 min/g wet wt perfusion with the same buffer containing type II collagenase enzyme (50 U/ml) (Worthington Biochemical). Excised ventricles were minced and triturated in storage solution containing (in mM) 70 KCl, 20 KH2PO4, 3 MgCl2, 10 HEPES, 10 glucose, 50 glutamic acid, 20 taurine, and 0.5 EGTA (pH 7.4). Isolated myocytes were suspended in storage solution and centrifuged twice (60g, 3 min), and the supernatant was discarded. The cells were cleaned during a 12-min incubation in warmed storage solution (37°C) containing 10 μg/ml of type IV protease (Sigma; St. Louis, MO) and 2 μg/ml of type IV DNAse (Sigma). The cells were centrifuged twice more at 60 g for 3 min with the final cell pellet resuspended in storage solution.

Isolation of ventricular myocytes for determination of cAMP was according to the following modification of the original isolation method (46). Isolated hearts were perfused as described in the previous isolation protocol. Myocytes were suspended in storage solution and allowed to settle for 20 min. The cell pellet was then resuspended for 20 min in storage solution containing 200 μM CaCl2, followed by resuspension in storage solution containing 600 μM CaCl2. The final cell pellet was resuspended in 3 ml of control “bath solution” containing (in mM) 133.5 NaCl, 4 KCl, 1.0 CaCl2, 12 NaH2PO4, 10 HEPES, 1.2 MgSO4, and 11.1 glucose (pH 7.4). Cells were evenly distributed among four microcentrifuge tubes before assay of cAMP. The pellet was resuspended in 0.1% sodium dodecyl sulfate for measurement of total protein content using bicinchoninic acid protein assay reagent kit (Pierce; Rockford, IL).

Ventricular membrane preparation.

Ligand binding, (−) [125I]iodocyanopindolol ([125I]ICYP) and isradipine ([3H]- PN200–110), was determined in ventricular membrane preparations. Isolated hearts from male and female rats were placed in cold (4°C) normal saline, and the left ventricle was dissected out. Wet weights were obtained on a combined batch of five left ventricles from male or five left ventricles from female hearts. Ventricles were then rapidly frozen in liquid nitrogen and stored at −80°C until the time of membrane preparation. To obtain the membrane fraction for [125I]ICYP binding experiments, a batch of five left ventricles (∼2 g wet tissue wt) from either sex were thawed and minced in 5 vol/g wet wt of ice-cold high-salt buffer composed of (in mM) 0.6 M KCl, and 10 Tris · HCl (pH 7.4) and the following protease inhibitors: aprotinin (1 μg/1 ml), leupeptin (1 μg/1 ml), and phenylmethylsulfonyl fluoride (0.5 mM). The tissue was homogenized by the use of Polytron (Brinkmann Instruments; Westbury, NY) for two 5-s bursts at a setting of 5. The homogenate was centrifuged at 4°C for 20 min at 5,000 g. The pellet was rehomogenized in ice-cold, low-salt buffer (6 mM HEPES, pH 7.4 with protease inhibitors) with two 3-s bursts (Polytron setting 5), followed by centrifugation at 4°C at 100,000 g for 60 min. The final pellet was resuspended in low-salt buffer containing protease inhibitors at a protein concentration of 200 μg/100 μl. Protein was determined by the bicinchoninic acid method (Sigma). Membranes used for the [3H]PN200–110 binding experiments were prepared from five left ventricles from either sex, thawed and minced in 5 vol/g wet wt of ice-cold (4°C) homogenizing buffer containing (in mM) 50 Tris · HCl, 3 MgCl2, 1 EDTA, and proteases (pH 7.4). The ventricles were homogenized with the use of Polytron for two 5-s bursts at a setting of 5. The homogenate was then centrifuged at 100,000 g for 60 min at 4°C. The resulting pellet was resuspended in homogenizing buffer at a protein concentration of 200 μg/100 μl. Total pellet protein content was determined by bicinchoninic acid assay (Sigma).

Myocyte shortening measurement.

Isolated ventricular myocytes were placed in a bath chamber, on the stage of an inverted microscope (Diaphot-TMD, Nikon), and allowed to attach to the glass bottom of the chamber. Myocyte shortening was measured in healthy, rod-shaped cells characterized by clear striations and no spontaneous contractile activity. Cells were continuously superfused with control bathing solution containing (in mM) 133.5 NaCl, 4.0 KCl, 1.0 CaCl2, 1.2 MgSO4, 10 HEPES, and 11.1 glucose (pH 7.4) and electrically paced with field stimulation (0.8 Hz, 12-ms duration, voltage intensity was twofold greater than that needed to elicit shortening). Unloaded cell shortening was measured with a video edge-detection system (51). An image of the cell was projected onto a television monitor by a charge-coupled device camera (TM-640 Sequential Scanning Camera, Pulnix). An oscilloscope monitored the signal emitted by cell edge movement, and a tracing of the signal was printed on a chart recorder (Dash II, Astromed; W. Warwick, RI). Maximal control (shortening in response to field stimulation and in the absence of drug) and isoproterenol-stimulated myocyte shortening was calculated from the chart recording and normalized to resting cell length.

Recording of whole cell Ca2+current.

Isolated myocytes were placed in an experimental bath chamber and allowed to attach to the glass bottom. The cells were superfused with the following bath solution (in mM): 133.5 NaCl, 4.0 CsCl, 1.0 CaCl2, 1.2 MgCl2, 11.1 glucose, and 10.0 HEPES, pH 7.4. Standard whole cell voltage clamp methods were used to recordI Ca (13), as routinely used in our laboratory (20). Patch pipettes with resistances of 2–4 MΩ were pulled from borosilicate glass capillary tubes with a two-stage pull technique, fire-polished, and filled with pipette solution composed of (in mM) 120 CsCl, 10 EGTA, 5 Na2ATP, and 10 HEPES, pH 7.2. The junction potential was electronically zeroed just before the formation of a seal between the pipette and the cell membrane. Cell membrane capacitance was estimated by integrating the area under the capacitative transient (generated by a 5-mV pulse) and by dividing by the voltage pulse. The L-type I Cawas elicited by 200 ms pulses to 0 mV from a holding potential of −80 mV (after a brief prepulse to −40 mV) at a frequency of 0.2 Hz. Prepulsing the membrane to −40 mV eliminated the Na+current (and any T-type Ca2+ currents). K+currents were eliminated by substitution of K+ in the bath and pipette solution with Cs+. ControlI Ca was measured in the absence of drug, and the isoproterenol-stimulated I Ca was measured in the presence of isoproterenol (1 μM) in the bath solution.

Current-voltage (I-V) curves were generated by increasing command potentials in 10 mV steps from −40 to +50 mV from a holding potential of −80 mV. Voltage-clamp protocols, data acquisition, and storage were accomplished by the use of pCLAMP software (version 5.1, Axon Instruments, Foster City, CA) and a digital-to-analog converter (Labmaster). Membrane currents were sampled at 5 KHz and stored on a computer. For I Ca analysis, currents were filtered at 2 KHz. I Ca was measured as the difference between the peak current and current at the end of the 200-ms pulse. The I Ca recorded in male and female myocytes was expressed as the I Ca density (I Ca normalized to cell capacitance).

To determine the effect of extracellular Ca2+ onI Ca in male and female myocytes, the L-typeI Ca was measured in the presence of 0.5, 1.0, 2.0, and 4.0 mM extracellular Ca2+, according to the whole cell voltage clamp methods as previously described. The controlI Ca was recorded in the presence of 1.0 mM CaCl2.

Radioligand binding studies.

β-Adrenergic receptor binding experiments were performed at 37°C in a final volume of 500 μl containing 100 μg of membrane protein, 50 mM MgCl2, 10 mM Tris · HCl (pH 7.4), and [125I]ICYP (0–220 pM) (specific activity 2,000 Ci/mmol). Nonspecific binding was determined in the presence of propranolol (1 μM) in duplicate sets of tubes. The reaction was terminated by rapid filtration through presoaked GF/B filters (0.3% polyethylenimine) (Whatman; Clifton, NJ) using a cell harvester (Brandel; Gaithersburg, MD). Filters were washed three times with 4 ml of ice-cold assay buffer. Bound [125I]ICYP was measured by gamma counting. Specific binding was determined by subtracting nonspecific binding from total binding. The dissociation constant (K d) and maximum binding capacity (Bmax) were determined using a nonlinear regression method for a one-site binding fit (Prism; GraphPad Software; San Diego, CA).

Dihydropyridine receptor binding experiments were performed under sodium lighting at 26°C in a final volume of 500 μl containing 200 μg membrane protein, 50 mM Tris · HCl, 3 mM MgCl2, 1 mM EDTA (pH 7.4) and tritiated [3H]PN200–110 (0–1.2 nM; specific activity 85.0 Ci/mmol). Nonspecific binding was determined with the addition of nifedipine (1 μM) to duplicate sets of tubes. The reaction was terminated by rapid filtration as described above. Bound [3H]PN200–110 was determined by liquid scintillation counting. The K d and Bmax were determined as previously described.

cAMP determination.

Isolated myocytes were incubated for 2 min in “bath solution” in the absence and presence of isoproterenol (1 μM). The cells were then centrifuged at a high speed (30 s; model 5415C, Eppendorf), and the supernatant was discarded. Ice-cold 65% ethanol (600 μl) was added to the cell pellet and mixed by vortexing. The cells were centrifuged again (20 min, high speed, 4°C, Eppendorf 5414C). The supernatant was stored in glass test tubes at 4°C until determination of cAMP content by a commercially available radioimmunoassay kit (Biomedical Technologies; Stoughton, MA). The cell pellet was resuspended in 1% sodium dodecyl sulfate for protein determination. The control and isoproterenol-stimulaled cAMP content was expressed as pmol cAMP/mg cell protein.

Drugs and materials.

All chemical compounds and drugs, except for radiolabeled receptor ligands, were from Sigma. [3H]PN200–110 and [125I]ICYP were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). All drug stock solutions and homogenizing and assay buffer solutions were prepared daily. A stock solution of 10−3 M isoproterenol was prepared in MilliQ water and stored in a dark, capped storage container to protect from light and air exposure and was used to prepare a final drug solution of 1 μM isoproterenol in bath solution. All responses to isoproterenol were measured in the presence of 1 μM of the drug. Propranolol, [125I]ICYP, and [3H]PN200–110 were prepared in their respective assay buffer solutions. A stock solution of 10−3 M nifedipine was prepared in dimethyl sulfoxide and used to make a final 10−6 M drug solution in assay buffer. [3H]PN200–110 and nifedipine solutions were prepared under sodium lighting.

Statistical analysis.

Values are expressed as the means ± SE for the following measures in female and male rats: 1) body weight, heart and left ventricular wet weights, cell length, and capacitance; 2) control and isoproterenol-stimulated myocyte shortening; 3) control and isoproterenol-stimulated peak I Ca;4) control and isoproterenol-stimulated cAMP content; and5) Bmax and K d for ICYP and [3H]PN200–110. Significant sex differences in rat and cell characteristics and the Bmax andK d from binding studies were determined by unpaired t-tests. Significant sex- and drug-dependent differences were determined by two-way ANOVA. Post hoc analysis to determine specific sex- and drug-dependent effects was determined by independent Student's t-tests. The level of significance was set at P ≤ 0.05 with Bonferroni correction when appropriate.


Female and male rat physical characteristics are presented in Table 1. Body and heart wet weights were not significantly different between males and females. Male myocytes were significantly longer than female myocytes. No significant difference was found in cell capacitance, an indirect measure of cell membrane area.

View this table:
Table 1.

Body weight, heart weight, and cell characteristics of myocytes isolated from male and female rats

Effect of isoproterenol on myocyte shortening.

Isoproterenol (1 μM) elicited a significant increase in myocyte shortening compared with control in both male and female cells (Fig.1 A) indicating β-adrenergic-mediated enhancement of myocyte contractile function in both sexes (P ≤ 0.01). Fractional shortening in the absence of drug tended to be greater in female than in male cells (9.7 ± 1.1 and 8.4 ± 0.6%, respectively); however, the difference was not significant. The isoproterenol-induced change in myocyte shortening (expressed as % change from control shortening) was significantly greater in males compared with females (P≤ 0.05) (Fig. 1 B). This indicates a sex difference in β-adrenergic-mediated enhancement of myocyte contractile function.

Fig. 1.

Effect of isoproterenol (1 μM) on female and male myocyte shortening. A: representative tracing of cell shortening in response to field stimulation in the absence of drug (control conditions) and in the presence of isoproterenol.B: isoproterenol-induced change in female and male cell shortening. Values are means ± SE; n = 26 cells from 9 female hearts and 24 cells from 8 male hearts. *P ≤ 0.05, male value is significantly different from female.

I-V relationship.

The relationship between membrane potential and Ca2+current (the I-V relationship) in the absence of isoproterenol was bell shaped as is typically found (Fig.2), and there was no significant difference between the male and female I-V relationship. The maximum current density occurred at 0 mV for both of the sexes. In the presence of 1 mM extracellular Ca2+, the maximal current density was −7.2 ± 0.7 pA/pF in females and the maximal current density was −6.2 ± 0.8 pA/pF in males.

Fig. 2.

Current-voltage (I-V) relationship for L-type Ca2+ current (I Ca) in male and female myocytes. The I Ca is normalized to cell capacitance (pA/pF). The current at each membrane voltage is the mean ± SE of 39 cells from 11 female hearts and 41 cells from 11 male hearts. For both sexes, peak I Ca was recorded at 0 mV. There were no significant differences in theI-V relationship.

Effect of isoproterenol on female and male myocyte Ca2+ current.

Figure 3 A shows superimposed Ca2+ current tracings from a male myocyte recorded in the presence and absence of isoproterenol (control conditions). The control L-type I Ca was significantly less in male myocytes compared with the I Ca recorded in female myocytes in the absence of β-adrenergic receptor stimulation (Fig. 3 B). The mean I Ca density in females was −5.6 ± 0.5 pA/pF, whereas in males, the meanI Ca density was −3.9 ± 0.6 pA/pF. Isoproterenol (1 μM) significantly increased the amplitude ofI Ca in both sexes. However, the magnitude of the drug-induced response was significantly greater in male compared with female myocytes (Fig. 3 C).

Fig. 3.

Effect of isoproterenol (1 μM) onI Ca in male and female myocytes. A: representative control (CON) and isoproterenol (ISO)-stimulated current trace in male myocyte. B: mean female and maleI Ca in the absence (open bars) and presence of isoproterenol (solid bars). C: percent isoproterenol-induced change I Ca in female and male myocytes. Values are means ± SE; n = 23 cells from 8 female hearts and 22 cells from 7 male hearts. #P ≤ 0.05, significantly different from respective no-drug control; *P ≤ 0.05, significantly different from female.

Effect of changes in extracellular Ca2+ on female and male myocyte Ca2+ current.

I Ca was examined in male and female myocytes in the presence of 0.5, 1.0, 2.0, and 4.0 mM extracellular Ca2+. At every concentration of extracellular Ca2+, a significantly greater I Cawas recorded in female myocytes compared with male myocytes (P ≤ 0.05) (Fig.4 A). However, the extracellular Ca2+-induced change in theI Ca (expressed as percent change fromI Ca measured at 1.0 mM extracellular Ca2+) was not significantly different between males and females (Fig. 4 B). In addition, no sex difference was found if the results were expressed as percentage of maximalI Ca (data not shown). This indicates that the relationship between the extracellular Ca2+ concentration and I Ca was not significantly different between the sexes.

Fig. 4.

Relationship between extracellular Ca2+ concentration and I Ca in female and male myocytes.A: mean I Ca density (pA/pF) recorded at 0 mV in female and male myocytes at 0.5, 1.0, 2.0, and 4.0 mM extracellular Ca2+. B: percent change inI Ca from current measured at 1.0 mM extracellular Ca2+. Values are means ± SE;n = 25 myocytes from 8 female rat hearts and 27 myocytes from 9 male rat hearts. *P ≤ 0.05, significantly different from male.

ICYP and PN200–110 binding.

Table 2 summarizes the results of β-adrenergic ([125I]ICYP) and Ca2+ channel ([3H]PN200–110) binding studies. The Bmax for the β-adrenergic ligand ICYP was twofold greater in male left ventricular membranes compared with females indicating a greater left ventricular β-adrenergic receptor density in males. The mean receptor density in males was 36.3 ± 7.0 fmol/mg protein compared with 18 ± 3.0 fmol/mg protein in females. No significant sex difference was found in the K d of specific ICYP binding to left ventricular membranes indicating no sex difference in left ventricular β-adrenergic receptor affinity.

View this table:
Table 2.

Female and male β-adrenergic and dihydropyridine receptor density and affinity

Left ventricular dihydropyridine receptor density (Bmax of [H3]PN200–110 binding) was significantly greater in females compared with males indicating a greater L-type Ca2+ channel abundance in female left ventricular membranes (Table 2). The affinity of [3H]PN200–110 for the dihydropyridine receptor was similar between females and males.

Effect of isoproterenol on myocyte cAMP production.

In both male and female myocytes, isoproterenol significantly increased cellular cAMP content (Fig. 5). However, the isoproterenol-induced change in cAMP content was twofold larger in males compared with females (P ≤ 0.05). In males, cAMP increased by 5.8 ± 1.1 pmol/mg protein, compared with 2.8 ± 0.8 pmol/mg protein in females.

Fig. 5.

Isoproterenol-induced change in cAMP content in female and male myocytes. Bars represent the isoproterenol-induced change in female and male myocyte cAMP content. Values are means ± SE;n = 10 batches of cells from 5 female and 5 male hearts respectively. *P ≤ 0.05, significantly different from female.


Short-term stimulation of myocardial function is primarily mediated by catecholamine-induced activation of the β-adrenergic signaling pathway. Our previous studies (37) demonstrated that the inotropic response to the β-adrenergic agonist isoproterenol is greater in the male rat atrium compared with the female rat atrium. The present study extends this finding to rat heart isolated ventricular cells. Here, we are the first to demonstrate that the isoproterenol-elicited increase in cell shortening is significantly greater in isolated male ventricular myocytes than drug-induced shortening in ventricular cells from female hearts. The finding implies an inherent sex difference in components of the β-adrenergic signaling pathway.

The results of our study revealed a twofold greater density of β-adrenergic receptors and greater isoproterenol-stimulated cAMP production in male myocytes compared with female myocytes. β-Adrenergic stimulation is known to result in cAMP/protein kinase A-induced phosphorylation the α1c-subunit of the L-type Ca2+ channel (19) and enhancement ofI Ca through these channels (32,41). Consistent with this, our results showed a greater isoproterenol-induced increase in I Ca in male myocytes. This suggests that there is enhanced β-adrenergic signaling in male myocytes compared with female myocytes. The possibility that a higher density of L-type Ca2+ channels in male myocytes could contribute to the larger positive inotropic response to β-adrenergic stimulation was ruled out by the results of the PN200–110 binding studies (Table 2). In fact, female myocytes had greater maximal binding of PN200–110 binding to the α1c-subunit of the channel than male myocytes suggesting higher Ca2+ channel density in females compared with males. This is consistent with the greater I Ca detected in females in the absence of isoproterenol (Fig. 3 B). The possibility that L-type Ca2+ channels may be functionally different between males than females cannot be ruled out; but this is unlikely based on the similar K d of PN200–110 binding and on the similarity of the Ca2+ I-V relationship in male and female myocytes (Fig. 2). Moreover, whereas the mean current density was greater in females than in males in the absence of drug (control conditions), the relationship between change in Ca2+ concentration and percent change inI Ca was the same (Fig. 4). This indicates that proportional increases in Ca2+ influx occurred with each change in extracellular Ca2+ and is more consistent with a difference in the number of Ca2+ channels than with a difference in channel conductance.

Several of the findings in this study would lead us to expect that in the absence of β-adrenergic stimulation (control conditions), cell shortening would be greater in female myocytes than male myocytes. First, female myocytes exhibited augmented I Cacompared with male myocytes at several extracellular Ca2+concentrations (Fig. 4). Second, when the Ca2+current/voltage relationship was determined, the peak current in females was somewhat larger than in males (Figs. 2 and 3 B). Finally, Ca2+ channel density was higher in female myocytes. Although our previous study (37) did find that contraction was greater in female than male atrium (37), this was not the case in the current study. While fractional shortening of female myocytes tended to be greater than male myocytes, the difference was not statistically significant. Several factors could explain this seemingly incongruent observation. One possibility is that the small sex difference in shortening observed in a single myocyte could summate in a multicellular preparation (such as the atrium or papillary muscle), thereby revealing comparatively greater contraction in female hearts. We are examining sex differences in contractile parameters in the rat papillary model to determine whether this is the case. In addition, contractile force or shortening is regulated by altering the delivery of free Ca2+ to the contractile proteins and by altering the sensitivity of contractile proteins to Ca2+ (38) and the mechanisms responsible for alterations in Ca2+ delivery and Ca2+sensitivity are multifactorial.

Of the many mechanisms that modulate delivery of Ca2+ to the contractile proteins, Ca2+ influx is only one. The amount of Ca2+ released from the sarcoplasmic reticulum that contributes to contraction for a given Ca2+ influx, depends on the concentration of Ca2+ in the sarcoplasmic reticulum and on the relationship between I Caand the probability of ryanodine receptor channel opening (9). Potentially, sarcoplasmic reticular Ca2+uptake via Ca2+-ATPase could be less efficient in females, thereby resulting in reduced Ca2+ uptake and storage. Sarcoplasmic reticular release mechanisms in female myocytes could also be less sensitive to trigger Ca2+. In support of this proposition, Bowling et al. (2) found that ovariectomy in rats produces a decrease in the K d of ryanodine binding that could be reversed by estrogen replacement. This observation suggests that estrogen may modulate a change in ryanodine receptor properties and indirectly suggests a potential for estrogen-mediated differences in Ca2+-stimulated Ca2+ release. Other regulators of Ca2+homeostasis could differ between males and females such as the Na+/Ca2+ exchanger. No other studies have examined sex differences these proposed mechanisms; therefore, their contribution to differences in Ca2+ homeostasis remains speculative.

The finding that the larger I Ca in female myocytes does not result in significantly greater cell shortening could also be related to sex differences in myofilament Ca2+sensitivity. A lower sensitivity in females would mitigate the effects of a greater Ca2+ influx. We (37) and others (42) have demonstrated that atria from female rat hearts are more sensitive to extracellular Ca2+ than atria from male hearts. But this cannot be extrapolated to suggest higher myofilament Ca2+ sensitivity in female myocardium. However, using skinned fibers from male and female atria, we were able to demonstrated enhanced responsiveness to Ca2+ in female atria compared with male atria. It is unknown whether ventricular myofilament Ca2+ sensitivity displays a sex difference. However, Wattanapermpool (43, 44) demonstrated that ovariectomy increased rat left ventricular myofilament Ca2+sensitivity and estrogen replacement prevented the increase in Ca2+ sensitivity. The finding suggests an influence of estrogen on cardiac muscle fiber sensitivity.

One limitation of this study is that it is impossible to use rats that are both weight and age matched. We used weight-matched male and female rats (Table 1), and therefore, there is an age disparity. Male rats were ∼12 wk old, and the females were ∼9 wk old. Both the males and females were sexually mature and not considered old (3). Capasso et al. (4) showed that there is no age-dependent difference in force or kinetics of papillary muscle contraction in this age range. In our previous studies, we used weight-matched animals to avoid issues of normalization of force measurements in atrial cardiac muscle. We continued using weight-matched rats in the current study based on the assumption that weight-matching the animals would increase the probability of obtaining a similar cell length between males and females. Interestingly, however, the average cell length was found to be longer in male myocytes, despite the finding that the cell membrane area (as measured by cell capacitance) was similar between the sexes. The two findings are not contradictory because length is a one-dimensional measure whereas capacitance encompasses three dimensions. However, because of these differences, all measures ofI Ca were normalized to take into consideration the membrane area of each cell, whereas the measure of cell shortening is expressed as fractional shortening to take resting cell length into consideration (9).

Few studies have examined sex differences in myocardial β-adrenergic signaling, but several investigations (7, 22, 25) have sought to determine whether sex hormones alter β-adrenergic receptors and response to β-adrenergic agonists. The results of these studies have been equivocal. We did not harvest the cells used in this study at any particular phase of the estrous cycle or attempt to examine the impact of sex hormones. Still, based on the recent identification of estrogen, androgen and progesterone receptors in cardiomyocytes (11, 12, 18, 21, 23, 26, 28, 33, 39), it could be speculated that the sex differences that we found are mediated by sex hormones.

In conclusion, this study is the first to demonstrate a sex difference in β-adrenergic-mediated enhancement of ventricular myocyte contractility and transsarcolemmal Ca2+ influx. Whereas myocytes from female hearts were shown to have a greaterI Ca in the absence of the drug, male myocytes exhibited a significantly greater isoproterenol-induced increase in the L-type I Ca and cell shortening. The results of this study also suggest that the mechanism responsible for this sex difference in response to β-adrenergic stimulation is, at least in part, a difference in the signal transduction pathway. Specifically, the maximal number of left ventricular β-adrenergic receptors and the isoproterenol-induced change in cAMP were greater in male compared with female myocytes. In addition, the mechanistic basis for the greater L-type I Ca in the absence of β-adrenergic stimulation in females is a higher Ca2+ channel density in the female cells. Functionally, these findings are in agreement with our previous report that demonstrated greater developed force in female rat atruim but an enhanced response to β-adrenergic stimulation in the male atrial myocardium.


The authors thank Dr. Jacquelyn Smith (Midwestern University) for conceptual and technical support.


  • This research was funded by National Research Service Award Grants NR-00073 (to D. Schwertz) and NR-70288 (to V. Vizgirda), by the Ralph and Marian Falk Medical Research Trust, by the Research Affairs Office of Midwestern University (to G. Wahler), and by the American Association of Critical Care Nurses.

  • Address for reprint requests and other correspondence: D. W. Schwertz, Univ. of Illinois, M/C 802, Depts. of Medical Surgical Nursing and Pharmacology, 845 S. Damen Ave., Chicago, IL 60612 (E-mail:Schwertz{at}uic.edu).

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


View Abstract