Sex differences in β-adrenergic responsiveness of action potentials and intracellular calcium handling in isolated rabbit hearts

Abstract

Cardioprotection in females, as observed in the setting of heart failure, has been attributed to sex differences in intracellular calcium handling and its modulation by β-adrenergic signaling. However, further studies examining sex differences in β-adrenergic responsiveness have yielded inconsistent results and have mostly been limited to studies of contractility, ion channel function, or calcium handling alone. Given the close interaction of the action potential (AP) and intracellular calcium transient (CaT) through the process of excitation-contraction coupling, the need for studies exploring the relationship between agonist-induced AP and calcium handling changes in female and male hearts is evident. Thus, the aim of this study was to use optical mapping to examine sex differences in ventricular APs and CaTs measured simultaneously from Langendorff-perfused hearts isolated from naïve adult rabbits during β-adrenergic stimulation. The non-selective β-agonist isoproterenol (Iso) decreased AP duration (APD90), CaT duration (CaD80), and the decay constant of the CaT (τ) in a dose-dependent manner (1-316.2 nM), with a plateau at doses ≥31.6 nM. The Iso-induced changes in APD90 and τ (but not CaD80) were significantly smaller in female than male hearts. These sex differences were more significant at faster (5.5 Hz) than resting rates (3 Hz). Treatment with Iso led to the development of spontaneous calcium release (SCR) with a dose threshold of 31.6 nM. While SCR occurrence was similar in female (49%) and male (53%) hearts, the associated ectopic beats had a lower frequency of occurrence (16% versus 40%) and higher threshold (100 nM versus 31.6 nM) in female than male hearts (p<0.05). In conclusion, female hearts had a decreased capacity to respond to β-adrenergic stimulation, particularly under conditions of increased demand (i.e. faster pacing rates and "maximal" levels of Iso effects), however this reduced β-adrenergic responsiveness of female hearts was associated with reduced arrhythmic activity.

Figure 1. Optical mapping of V_m and Ca²⁺ in isolated rabbit hearts.

Left, Langendorff-perfused isolated rabbit heart mounted in the tissue bath with the anterior surface of the ventricles centered in the viewing window. Center, heart illuminated with the high power green (530 nm peak λ) LEDs with the black box representing the field of view. Right, representative recordings of the volume-conducted electrocardiogram (ECG), optical AP (V_m), and CaT (Ca²⁺) measured simultaneously during a single paced beat.

Figure 2. Baseline sex differences in steady-state ventricular APs and CaTs.

Summary data for action potential duration (APD₉₀, ms), calcium transient duration (CaD₈₀, ms) and the time constant of calcium recovery (τ, ms) at 3 and 5.5 Hz pacing at either the LV base (left) or apex (right) in the absence of β-adrenergic stimulation (baseline). *p<0.05, female vs male.

Figure 3. Less Iso-induced decrease of APD₉₀ in female hearts.

A) Representative APs at baseline (green traces) and during treatment with Iso (316.2 nM, orange traces) normalized and superimposed to demonstrate the sex difference in Iso effects during pacing at 3 Hz (top) and 5.5 Hz (bottom). B) Summary data for the decrease in APD₉₀ (versus baseline values) as a function of the dose of Iso (1, 10, 31.6, 100, 316.2 nM) in the LV base (left) and apex of female (n = 5) and male (n = 5) hearts during pacing at 3 Hz (top) and 5.5 Hz (bottom). *p<0.05, female versus male.

Figure 4. No significant sex differences in Iso-induced decrease of CaD₈₀.

A) Representative CaTs at baseline (green traces) and during treatment with Iso (316.2 nM, orange traces) normalized and superimposed to demonstrate the Iso-induced shortening of CaD₈₀ at both 3 Hz (top) and 5.5 Hz (bottom) pacing. B) Summary data for the decrease in CaD₈₀ (versus baseline values) as a function of the dose of Iso (1, 10, 31.6, 100, 316.2 nM) in the LV base (left) and apex (right) with pacing at 3 (top) and 5.5 Hz (bottom) for female (n = 5) and male (n = 5) hearts. Female versus male, p = NS for all comparisons.

Figure 5. Less Iso-induced decrease of τ in female hearts.

A) Relaxation phase of the CaT (peak amplitude to full recovery to diastolic levels) at baseline and during treatment with Iso superimposed to demonstrate the sex differences in the effect of Iso on the CaT decay kinetics at both 3 Hz (top) and 5.5 Hz (bottom) pacing. Single exponential decay functions (blue curves) were fit to the relaxation phase of the CaT, with the time constant of decay (τ) shown for each trace. B) Sex differences in the decrease in τ (versus baseline values) as a function of the dose of Iso (1, 10, 31.6, 100, 316.2 nM) in the LV base (left) and apex (right) of female (n = 5) and male (n = 5) hearts with pacing at 3 (top) and 5.5 Hz (bottom). *p<0.05, female versus male.

Figure 6. Iso-induced SCR and ectopic activity.

A) Representative CaTs from a male heart during the last three paced beats (S1) of a 3 Hz drive train. Top, at baseline calcium levels return to diastole. During treatment with Iso (31.6 nM), an SCR occurs shortly after the return to diastolic calcium levels. B) Sex differences in the frequency of pacing protocols that elicited SCRs and EBs during treatment with Iso. C) Simultaneous recordings of Ca²⁺, V_m, and the volume-conducted ECG from a male heart showing the last three paced beats (S1) followed by an SCR (arrow) and an EB elicited by treatment with 31.6 nM Iso. Note that the smaller, regularly occurring activations (#) in the ECG are derived from the intrinsic activity of the left atrium. This confirms the complete atrioventricular dissociation in CHB hearts. D) Sex difference in the coupling interval (ms) between the activation of the last paced beat and EBs elicited during treatment with Iso. *p<0.05, female vs male.