Effect of oral hormonal contraceptive pill use on the hemodynamic response to the cold pressor test

Abstract

Acute increases in sympathetic nervous system activity (SNA) often elicit peripheral vasoconstriction and increases in blood pressure (BP). Given sympathetic support of BP is modulated by ovarian sex hormones (e.g., estradiol), we sought to examine the effect of menstrual cycle and oral hormonal contraceptive pill (OC) phase on the hemodynamic response to acute increases in SNA. We hypothesized sympathoexcitation via cold pressor test (CPT) would elicit greater peripheral vasoconstriction and increases BP in females with natural menstrual cycles (NC) compared with females taking OC. We further hypothesized that SNA-mediated vasoconstriction would be attenuated during the high estradiol (HE) phase versus the low estradiol (LE) phase of the menstrual/pill cycle. Female NC (n = 11, 25 ± 1 yr) and OC (n = 10, 24 ± 1 yr) participants were studied during the LE (early follicular, placebo pill) and HE (late follicular, active pill) phase of the menstrual/pill cycle. BP (finger photoplethysmography), heart rate (HR, ECG), and forearm blood flow (FBF, venous occlusion plethysmography) were measured during a 5-min baseline and a 2-min CPT. CPT elicited an increase in BP in both groups (time, P < 0.01). During CPT, OC participants exhibited greater and sustained increases in HR compared with NC participants (group × time, P < 0.01). Higher HRs were met with increases in FBF in OC participants during the CPT, which was not observed in NC participants (group × time, P < 0.01). OC participants exhibit greater increases in HR, and paradoxical vasodilation during acute sympathetic activation compared with NC participants. Group differences are unaffected by menstrual/pill phase.

NEW & NOTEWORTHY Acute increases in sympathetic nervous system activity often elicit peripheral vasoconstriction and increases in blood pressure (BP). Given sympathetic support of BP is modulated by ovarian sex hormones (e.g., estradiol), we sought to examine the effect of menstrual cycle and oral hormonal contraceptive pill (OC) phase on the hemodynamic response to acute increases in sympathetic nervous system activity via the cold pressor test. We show OC participants exhibit paradoxical vasodilation during acute sympathetic activation compared with participants with natural menstrual cycles; notably, group differences were unaffected by menstrual/pill phase.

INTRODUCTION

Individuals with high blood pressure (BP, i.e., hypertension) are at an increased risk of cardiovascular and all-cause mortality (1). The prevalence of hypertension is lower among premenopausal adult females compared with age-matched males (2). However, hypertension prevalence increases exponentially in females as they age (2). The exponential increase in blood pressure in older adult females coincides with the time of menopause and reduced secretion of ovarian sex hormones (i.e., estradiol) (3).

The sympathetic nervous system is an important regulator of blood pressure (4). Postmenopausal females exhibit greater sympathetic support of blood pressure when compared with premenopausal females (5, 6). Furthermore, the transduction of sympathetic nervous system activity (SNA) into vascular tone is greater in postmenopausal than in premenopausal females (7). These data suggest the loss of circulating ovarian sex hormones may alter the relationship between sympathetic nervous system activity and peripheral vascular tone. In support of this idea, in premenopausal females, the vasoconstrictor response to sympathetic activation changes in the presence and/or absence of estradiol and may be dependent upon hormone source.

Data from Minson et al. (8) show female participants exhibit higher muscle sympathetic nerve activity (MSNA) and plasma norepinephrine during the high hormone phase (midluteal) compared with the low (early follicular) hormone phase of the natural menstrual cycle (NC), yet vascular resistance remains unchanged. In contrast, MSNA is unaffected by oral hormonal contraceptive pill (OC) phase; however, blood pressure and vascular resistance tend to be lower during the high hormone phase compared with the low hormone phase (9). Although controversy exists (10), exogenous estradiol supplementation may attenuate vasoconstrictor responses to norepinephrine and enhance basal nitric oxide release (11, 12). Together these data may support an attenuated vascular response to sympathetic nervous system activity in the presence of high versus low circulating levels of ovarian sex hormones.

With this information in mind, we used fluctuations in estradiol during the natural menstrual cycle (NC) and oral hormonal contraceptive pill phase (OC) to isolate the influence of low and high concentrations of estradiol on the vascular response to sympathetic activation while mitigating the confounding effects of progesterone. We hypothesized female participants taking OCs would exhibit less skeletal muscle vasoconstriction in response to sympathetic activation compared with NC participants. We further hypothesized sympathetically mediated vasoconstriction within the skeletal muscle vasculature would be attenuated in both groups during the high estradiol compared with the low estradiol phase of the menstrual/pill cycle.

METHODS

Participants

All experiments were approved by the Institutional Review Board at the University of Missouri (IRB No. 2011312) and conformed to the ethical principles of the Declaration of Helsinki, including registration in a database (NCT04436731). Data from a subset of participants with natural menstrual cycles were published previously testing hypotheses unrelated to the present investigation (13). Participants were recruited from Columbia, MO, and surrounding areas using advertisements and word of mouth. All participants reported being female, premenopausal, nonobese (body mass index, <30 kg/m²), nonnicotine users between the ages of 18–45 yr with blood pressure <140/90 mmHg, and taking no medications known to affect autonomic or cardiovascular function (other than oral hormonal contraceptive pills). Before participation, individuals provided written informed consent.

All participants completed two study visits, one during the low estradiol phase of the menstrual (early follicular, days 2–6) or pill (placebo pill, days 4–7) cycle, and one during the high estradiol phase of the menstrual (late follicular, determined using commercially available ovulation kit) or pill (active pill, days 14–19) cycle. For simplicity, the term high estradiol refers to both high endogenous (late follicular) and high exogenous (active pill) estradiol. First-generation oral hormonal contraceptive pills were excluded because high dosage of ethinyl estradiol (>50 μg) may elicit adverse vascular effects (14). Therefore, inclusion criteria for participants taking oral hormonal contraceptives included second, third, and fourth-generation conventional (28-day length) monophasic oral contraceptive pills. Women were required to have been consistently taking their prescribed formulation for ≥0.5 year (range, 0.5–5.0 years), determined by self-report. See Table 1 for details regarding oral hormonal contraceptive pill formulation and concentration.

Table 1.

Oral hormonal contraceptive use

Count	Generation	Brand	Progesterone Formulation	Dose, mg
				Estradiol	Progesterone
1	2	Aviane/Falmina	Levonorgestrel	0.020	0.10
2	3	Estarylla	Norgestimate	0.035	0.25
2	3	Sprintec	Norgestimate	0.035	0.25
1	3	Femynor	Norgestimate	0.035	0.25
2	3	Enskyce	Desogestrel	0.030	0.15
1	3	Mircette	Desogestrel	0.020	0.15
1	4	Loryna/Gianvi	Drospirenone	0.020	3.00

Data are reported from n = 10 female participants taking oral hormonal contraceptives.

Participants refrained from caffeine, strenuous exercise, alcohol, and nonsteroid anti-inflammatory agents for 24 h before their scheduled study started. Participants arrived at the laboratory after a 4-h fast (no food or drink other than water). At the start of each study visit, participants’ height, weight, and vital signs (blood pressure and heart rate) were assessed. All participants were required to have a negative pregnancy (urine) test. Participants quietly rested supine for 15 min, after which a 40 mL blood sample was taken via venipuncture. Blood samples were centrifuged, and serum was stored at 4°C before assessment of estradiol, progesterone, luteinizing hormone, and total testosterone (immunoassay; Quest Diagnostics Laboratories, Columbia, MO). For measures of norepinephrine and epinephrine, samples were centrifuged and plasma was stored at −80°C before assessment (high-performance liquid chromatography; Quest Diagnostics Laboratories, Columbia, MO).

Instrumentation

Participants were instrumented with a three-lead electrocardiogram to measure heart rate (Lead II, Bio Amp FE132, ADinstruments, Colorado Springs, CO) and finger photoplethysmography for blood pressure (Human NIBP Controller ML282, ADinstruments, Colorado Springs, CO), which was calibrated to upper arm blood pressure (automatic sphygmomanometer) taken in triplicate. Forearm blood flow (FBF) was assessed via venous occlusion plethysmography on the participant’s left arm (D.E. Hokanson Inc., Bellevue, WA). To do so, a cuff was placed around the wrist and inflated to 220 mmHg to exclude hand circulation from the measurement. A second cuff was placed around the upper arm, cyclically inflated to 50 mmHg for 7 s, and then deflated (0 mmHg) for 8 s to obtain one blood flow measurement every 15 s (15). Individuals wore a mask covering the nose and mouth for the entirety of the data collection period to monitor respirations. All testing was done in the supine position in a temperature-controlled room (16).

Protocol

Following instrumentation, participants underwent a 5-min quiet resting period, followed by a cold pressor test (CPT). The CPT is a well-established sympathoexcitatory maneuver used to assess α-adrenergic receptor-mediated vasoconstriction (17–20). The magnitude of sympathetic activation and blood pressure response to CPT have been shown to be reliable/reproducible (21, 22). During the CPT, participants had their left foot passively immersed up to the ankle in an ice-water bath maintained at 0°C–4°C for 2 min to acutely activate the sympathetic nervous system (20). After removal of the foot from the ice water, participants were asked to rank perceived thermal (cold) and pain severity using a modified Borg scale (23).

Data Analysis

Hemodynamic variables were collected continuously during the 5-min of quiet rest and 2-min CPT. Stroke volume was estimated from the finger blood pressure waveform using the Modelflow method (LabChart, ADinstruments), which incorporates age and sex, and cardiac output and total peripheral resistance (TPR) were calculated. The Modelflow method has been validated in a variety of populations and experimental protocols and has been shown to reliably track fast changes in hemodynamics (24–27). Hemodynamic data were analyzed beat by beat and then averaged individually for each participant across 15-s intervals. Measures of forearm blood flow (FBF) are expressed as milliliters of blood/100 mL of forearm volume/min and are reported from an average of approximately three to four total flows. FBF was normalized for mean arterial blood pressure to determine forearm vascular conductance (FVC; forearm blood flow ÷ mean arterial blood pressure × 100 mmHg) and is expressed as milliliters of blood/100 mL of forearm volume/100 mmHg/min. Forearm vascular resistance (FVR) was also calculated (mean arterial blood pressure ÷ FBF ÷ 100). Due to equipment issues, blood flow data were unavailable from one participant in the NC group and thus FBF, FVC, and FVR are reported from n = 10. Changes in hemodynamic variables from baseline were calculated (CPT − baseline) and are expressed as a relative (%) change [(CPT − baseline) ÷ baseline × 100].

Statistical Analysis

Participant demographics were compared between groups using an unpaired t test. Blood samples and resting hemodynamics were assessed using a two-way mixed-effects analysis of variance. Post hoc multiple comparisons were made using Šídák’s multiple comparisons test. Main outcome variables included FVC and TPR. Comparisons between group (NC/OC) and phase (low/high) on the effect of CPT (time) were conducted using a three-way mixed-effects analysis of variance with Geisser–Greenhouse correction. An α < 0.05 was considered statistically significant. Data are reported as means ± SD.

RESULTS

Participant Characteristics

Groups did not differ by age (NC: 25 ± 4, OC: 24 ± 3 yr; P = 0.55), height (NC: 165 ± 4, OC: 164 ± 5 cm; P = 0.39), weight (NC: 62 ± 6, OC: 56 ± 7 kg; P = 0.08), nor body mass index (NC: 23 ± 2, OC: 21 ± 2 kg/m²; P = 0.15). There was a significant interaction between group and phase for estradiol and progesterone (P < 0.01; Table 2). Post hoc analysis showed endogenous estradiol (P = 0.78) and progesterone (P = 0.99) were not different between the high and low hormone phases in OC participants. In contrast, NC participants exhibited higher estradiol (P < 0.01) and progesterone (P < 0.01) during the high hormone phase compared with low hormone phase of the menstrual cycle. Post hoc analysis revealed that estradiol (P < 0.01) and progesterone (P < 0.01) were greater in NC participants compared with OC participants during the high hormone phase of the menstrual/pill cycle, despite no difference during the low hormone phase (estradiol: P = 0.97; progesterone: P = 0.96).

Table 2.

Resting hemodynamics

	Natural Menstrual Cycles		Oral Hormonal Contraceptives		P Value
	Low	High	Low	High	Group	Phase	Interaction
Participant characteristics
Testing day	3 ± 1	16 ± 2†	5 ± 1*	17 ± 2†	0.02	<0.01	0.36
Progesterone, ng/mL	0.56 ± 0.07	1.02 ± 0.19†	0.53 ± 0.02	0.53 ± 0.02*	<0.01	<0.01	<0.01
Estradiol, pg/mL	28 ± 12	171 ± 140†	35 ± 15	17 ± 5*	<0.01	<0.01	<0.01
Testosterone (total), ng/dL	28 ± 9	41 ± 12†	39 ± 16	29 ± 13†	0.55	0.44	<0.01
Luteinizing hormone, mIU/mL	4 ± 2	11 ± 11†	4 ± 3	2 ± 3*	0.052	0.059	<0.01
Norepinephrine, pg/mL	221 ± 199	184 ± 36	200 ± 74	215 ± 88	0.72	0.43	0.59
Resting hemodynamics
Mean arterial blood pressure, mmHg	80 ± 7	78 ± 7	79 ± 5	78 ± 8	0.86	0.11	0.46
Heart rate, beats/min	68 ± 12	69 ± 8	70 ± 10	75 ± 7	0.27	0.065	0.16
Stroke volume, mL/beat	51 ± 8	51 ± 6	48 ± 6*	44 ± 7*	0.04	0.19	0.17
Cardiac output, L/min	3.2 ± 0.6	3.3 ± 0.2	3.2 ± 0.5	3.1 ± 0.6	0.61	0.99	0.64
Total peripheral resistance, mmHg·s/mL	1.7 ± 0.4	1.5 ± 0.2	1.6 ± 0.3	1.7 ± 0.3	0.67	0.53	0.19
Forearm blood flow, mL/min/dL	2.4 ± 0.8	2.6 ± 0.8	2.0 ± 0.6	2.6 ± 0.7	0.44	0.39	0.08
FVC, mL/min/dL/100 mmHg	3.2 ± 0.9	2.9 ± 0.8	2.5 ± 0.9	3.1 ± 0.8	0.49	0.55	0.04
FVR, 100 mmHg/mL/min/dL	0.34 ± 0.10	0.38 ± 0.14	0.44 ± 0.12	0.34 ± 0.08	0.55	0.31	<0.01

Values are means ± SD; n = 11 [natural menstrual cycle (NC)] and n = 10 [oral hormonal contraceptive (OC)], unless otherwise noted [NC: forearm blood flow (FBF)/forearm vascular conductance (FVC) n = 10, estrogen/progesterone/testosterone n = 8, norepinephrine n = 7; OC: estrogen/progesterone n = 10, testosterone/norepinephrine n = 9]. FVR, forearm vascular resistance. Testing day refers to the number of days from the start of the self-report menstrual or pill cycle. Results were compared using a two-way mixed-effects analysis of variance. Post hoc multiple comparisons were conducted using Sidak's multiple comparisons test. *P < 0.05 vs. NCs. †P < 0.05 vs. low.

Plasma norepinephrine concentrations did not differ by group (group, P = 0.72) nor phase (phase, P = 0.43). Similarly, resting mean arterial blood pressure, heart rate, cardiac output, TPR, FBF, and FVC did not differ between groups or phases (all, P > 0.05). OC participants exhibited lower SV at baseline compared with NC participants (group, P = 0.04), which was unaffected by menstrual/pill phase (phase, P = 0.19). See Table 2.

Hemodynamic Response to Sympathetic Activation

Sympathetic activation via CPT resulted in a time-dependent increase in mean blood pressure (time, P < 0.01; Fig. 1A), due primarily to an increase in cardiac output (time, P < 0.01; Fig. 1B). The time-dependent increase in cardiac output during CPT differed by group (group × time, P = 0.03), attributed to group differences in the rise in heart rate (group × time, P < 0.01; Fig. 1C) rather than stroke volume (group × time, P = 0.86; Fig. 1D). Although the TPR response to CPT did not differ by group (group, P = 0.49) or phase (phase, P = 0.71; Fig. 1E), group differences were observed within the skeletal muscle vasculature (FBF: group, P < 0.01; FVC: group, P = 0.03; Fig. 2, A and B). OC participants exhibited an increase in FBF and FVC during CPT, which was not observed in NC participants (FBF: group × time, P < 0.01; FVC: group × time, P = 0.01). The FVR response to CPT was unaffected by group and/or phase (all P > 0.05; Fig. 2C). There was no effect of group or phase on the perception of cold (P = 0.56 and P = 0.65) nor pain (P = 0.052 and P = 0.65) during the CPT (data not shown).

Figure 1.

Systemic hemodynamic response to cold pressor test. Data are reported from natural menstrual cycle (NC): n = 11; oral hormonal contraceptive (OC): n = 10. Results were compared using a three-way mixed-effects analysis of variance with Geisser–Greenhouse correction and matched by phase and time, with α = 0.05. Significant main effects and interactions are noted.Shown are mean blood pressure (A), cardiac output (B), heart rate (C), stroke volume (D), and total peripheral resistance (E).

Figure 2.

Skeletal muscle vascular response to cold pressor test. Data are reported from natural menstrual cycle (NC): n = 10; oral hormonal contraceptive (OC): n = 10. Results were compared using a three-way mixed-effects analysis of variance with Geisser–Greenhouse correction and matched by phase and time, with α = 0.05. Significant main effects and interactions are noted. Shown are forearm blood flow (A), forearm vascular conductance (B), and forearm vascular resistance (C).

DISCUSSION

Present novel findings show the increase in blood pressure during acute sympathetic activation does not differ between NC and OC participants; however, contributing mechanisms are divergent. We show that participants taking OCs exhibit local skeletal muscle vasodilation in response to acute sympathetic activation (Fig. 2B). Furthermore, OC participants exhibit greater and sustained increases in heart rate (Fig. 1C) compared with NC participants. Together these novel findings highlight differences between endogenous (NC) and exogenous (OC) estradiol on the integrated cardiovascular response to acute sympathetic activation.

Acute increases in sympathetic nervous system activity result in postsynaptic release of neurotransmitters (i.e., norepinephrine), which bind to α-adrenergic receptors on the vascular smooth muscle, promoting vasoconstriction and an increase in blood pressure. Following menopause and the loss of endogenous estradiol, sympathetic support of blood pressure (5, 6), and the transduction of sympathetic nervous system activity into vascular tone (7) is increased. These data support the effect of circulating ovarian sex hormones on the relationship between sympathetic activity and peripheral vascular tone. Indeed, the amount of vascular resistance achieved per amount of sympathetic nervous system activity may be greater during the low, versus high, hormone phase of a natural menstrual cycle (8) and this difference may be exaggerated with oral hormonal contraceptive use (9).

The CPT is a well-established sympathoexcitatory maneuver used to assess α-adrenergic receptor-mediated vasoconstriction (17–20). Importantly, prior work has shown the magnitude of muscle sympathetic nervous system activity in response to the CPT is unaffected by hormone phase and/or oral hormonal contraceptive pill use (28). Similarly, the blood pressure response to a hypotensive stimulus was also shown to be no different between NC and OC participants and was unchanged across the menstrual/pill cycle (29–31). Although there was no main effect of group on the blood pressure response to CPT, we observed a group-by-phase interaction (Fig. 1A), suggesting within groups there was a differential effect of hormone concentrations that warrants further investigation. Despite this lack of inherent between-group difference in the blood pressure response to CPT, contributing mechanisms were divergent.

In NC participants, we observe a ∼20% reduction in FVC during the second minute of the CPT using the foot (Fig. 2B), which has been consistently observed in prior research (32–34). In contrast, we show for the first time that OC participants exhibit paradoxical skeletal muscle vasodilation in response to acute sympathetic activation. When examining sex- and age-related differences in sympathetic response to the CPT, Miller et al. (35) found a subset of young, premenopausal female participants increased femoral artery blood flow during the CPT. On closer inspection of the study cohort, 7 of the 12 female participants studied reported taking oral hormonal contraceptive pills. Although Miller et al. did not control for menstrual and/or pill cycle phase, present data suggest the type of hormone (endogenous/exogenous) rather than the phase of the menstrual cycle (low/high estradiol) may be more important in the peripheral vascular response to sympathetic activation.

We further show OC participants exhibit greater and sustained increases in heart rate (Fig. 1C) compared with NC participants. We speculate higher heart rates may be a compensatory mechanism to maintain blood pressure in the context of peripheral vasodilation. Alternatively, higher heart rates in OC participants could be a preceding event, ultimately facilitating the increase in vascular conductance (36). Similarly, greater cardiac output has been observed during postural changes in OC participants compared with NC participants (37). The authors speculated group differences may be due to enhanced peripheral vasodilation and/or reduced sympathetically mediated vasoconstriction in OC participants relative to NC participants (37), interpretations which are supported by present data.

Potential Mechanisms

Mechanisms contributing to vasodilation in response to sympathetic activation in participants taking OCs are likely multifactorial and beyond the scope of the present investigation. However, prior work has shown that, relative to NC, adult females taking OCs exhibit augmented β-adrenergic mediated skeletal muscle vasodilation (38) and greater basal nitric oxide production and release (39). Given β-adrenergic mediated vasodilation may offset α-adrenergic mediated vasoconstriction (40) in response to sympathetic activation, OC use may enhance the ability to oppose sympathetically mediated vasoconstriction relative to NC. This is supported by our data (Fig. 2B) demonstrating an increase in FVC in response to sympathetic activation in OC participants. The synthetic estrogen found in OCs (17-α ethinyl estradiol) exhibits 6–10 times more estrogenic activity relative to endogenous, 17-β estradiol. Mechanistic data from Riedel et al. (42) observed vessels exposed to estradiol increased β₁- and β₃-adrenergic receptor mRNA expression in the vascular endothelium as well as increased receptor sensitivity to pharmacologic agonists. Thus, it is reasonable to speculate exogenous estradiol supplementation may upregulate both vascular and cardiac β-adrenergic receptors, contributing not only to greater skeletal muscle vasodilation but also to higher heart rates and cardiac output observed presently. Whether these adaptations to exogenous estradiol translate to females following menopause is currently inconclusive (43, 44).

It is also worth considering the confounding and/or antagonizing influences of testosterone and progesterone on present findings. Although our understanding in female adults is limited, testosterone may augment the blood pressure response to CPT in males (45). Progesterone may also reduce vasodilatory capacity (46) and augment sympathetic tone (47). Given testosterone and progesterone are higher during the high estradiol phase in the NC versus OC group (Table 2), the potential contribution of these changes on present findings should not be overlooked.

Experimental Considerations

Strengths of the present investigation include a robust study cohort of human research participants tested in random order during distinct time points of the menstrual/pill cycle with blood samples and ovulation tests confirming adequate scheduling of study visits. We show greater circulating estradiol during the high estradiol phase in NC participants compared with OC participants, supporting effective timing of study visits for the NC group, as well as exogenous suppression of endogenous hormones by OC use.

Despite these strengths, there are a few limitations that should be acknowledged. First, we do not have direct quantification of MSNA. Notably, baseline norepinephrine concentrations did not differ by group, which is consistent with no difference in levels of resting sympathetic activity and agrees with previously published studies (9, 48). Prior work has also shown the sympathetic response to CPT is reproducible within and across study visits (21, 22) and does not differ between NC and OC participants (28), although much of this work has been conducted using CPT of the hand, rather than the foot. Second, although Modelflow has been validated using Doppler ultrasound (27, 49) in a variety of study conditions (50), caution should be applied in interpretation during large changes in peripheral resistance (50).

Finally, baseline blood pressures did not differ by group (Table 2). These data disagree with prior work supporting higher resting blood pressures in females taking OCs compared with NC adults (48), and a duration-dependent increase in the risk of hypertension associated with OC use (51). Discrepancies may be due to a smaller sample size in the present investigation. Furthermore, the duration of OC use in the present study was relatively short (2.7 ± 1.8 yr), and rigorous screening ensured current study participants were young and otherwise healthy. It is reasonable to propose a longer duration of OC use may potentiate the cardiovascular response to sympathetic activation.

Perspectives and Conclusion

Present findings show differences in the hemodynamic response to sympathetic activation in NC and OC participants, which may be attributed to differential effects of endogenous (NC) versus exogenous (OC) estradiol on the cardiovascular system. Results support the importance of considering OC use as a biological variable in cardiovascular research and echo the sentiment of previously published work highlighting the critical need for a comprehensive study of the vascular implications of OC use (52). As elevated sympathetic nervous system activity and the blood pressure responses to stress are predictive of the development of hypertension (53), better understanding the influence of estradiol (both endogenous and exogenous) on blood pressure and peripheral vascular function is clinically pertinent in the pathophysiology of hypertension in adult females.

GRANTS

This work was funded by National Heart, Lung, and Blood Institute Grants HL130339 and HL153523 (to J.K.L.) and American Heart Association Grant AHA 909014 (to D.W.J.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

D.W.J. and J.K.L. conceived and designed research; D.W.J., J.L.H., and J.K.L. performed experiments; D.W.J., A.M.V., and J.L.H. analyzed data; D.W.J., A.M.V., and J.K.L. interpreted results of experiments; D.W.J. and A.M.V. prepared figures; D.W.J. drafted manuscript; D.W.J., A.M.V., J.L.H., and J.K.L. edited and revised manuscript; D.W.J., A.M.V., J.L.H., and J.K.L. approved final version of manuscript.