Intracellular β1-Adrenergic Receptors and Organic Cation Transporter 3 Mediate Phospholamban Phosphorylation to Enhance Cardiac Contractility

Rationale: β 1 -adrenoceptors (β 1 ARs) exist at intracellular membranes and Organic Cation Transporter 3 (OCT3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β 1 AR in cardiac contractility remains to be elucidated. Objective: Test localization and function of intracellular β 1 AR on cardiac contractility. Methods and Results: Membrane fractionation, super-resolution imaging, proximity ligation, co-immunoprecipitation and single-molecule pulldown demonstrated a pool of β 1 ARs in mouse hearts that was associated with sarco/endoplasmic reticulum Ca 2+ -ATPase at the sarcoplasmic reticulum (SR). Local protein kinase A (PKA) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared to wild type (WT), myocytes lacking OCT3 (OCT3KO) responded identically to the membrane-permeant βAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3KO selectively suppressed calcium transients and contraction responses to norepinephrine, but not isoproterenol. Furthermore, sotalol, a membrane-impermeant βAR-blocker suppressed isoproterenol-induced PKA activation at the PM, but permitted PKA activation at the SR, phospholamban phosphorylation and contractility. Moreover, pretreatment with sotatol in OCT3KO myocytes prevented norepinephrine induced PKA activation at both PM and the SR and contractility. Conclusions: Functional β 1 ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular β 1 ARs requires catecholamine transport via OCT3.

Abstract 383: Cavβ2a Transgenic Mouse as a Hfpef Model With Hypercontractile Cardiomyocytes

Objective: Heart failure (HF) with preserved ejection fraction (HFpEF) is characterized by a preserved cardiac EF, the presence of HF symptoms and diastolic dysfunction. There is a lack of animal models for exploring the mechanisms and treatments of HFpEF. This study aimed to test if cardiomyocyte (CMs) specific, inducible Cavβ2a transgenic (Cavβ2a TG) mice, having hypercontractile CMs, could be a model for HFpEF. Methods: High (HE) and low (LE) expression Cavβ2a TG mice were studied since transgene expression at the age of 2m till the age of 8m monthly to evaluate the systolic and diastolic function. At 8m, animals were euthanized for intra-left ventricular hemodynamic measurement, myocyte function and histological analyses, and Western blotting measurements. Results: LE and HE TG ventricular myocytes (VMs) had greater Ca2+ currents at the age of 2m (LE increased by 95.5%; HE increased by 171.9% ) and maintained at a similar level at the age of 8m. VM contraction and calcium transients had greater amplitudes in TG than in control VMs while the time from the peak contraction to 90% relaxation and the tau of Ca2+ transient decay of VMs were not different between groups probably due to enhanced NCX expression and increased SERCA activity because of increased phospholamban phosphorylation at the Thr17 site. Till 8m, LE and HE mice had a higher level of mortalities than control mice (LE: 32%, HE: 15%, control:5%), but more HE (87.5%) mice showed pleural effusion. The EF was highest in LE mice at the age of 2m but decreased to the same (>60%) as in HE and control mice at 8m. The ratio of mitral E to mitral inflow A wave velocities (E/A) was increased significantly at 3 and 4m then decreased to the lowest in HE at 5m followed by a rise at 6-8m, while LE mice had the lowest E/A at 5m and 6m and increased at 8m. E wave to mitral annulus e’ velocity (e’ wave) was higher in LE and HE groups than control mice. More fibrosis of the LV tissue, cardiomyocyte necrosis was observed in HE than in LE hearts. LE and HE mice had greater left ventricular wall relaxation rates, as indicated by larger reversed radial and longitudinal strain rates. Conclusion: LE and HE mice showed progression of diastolic dysfunction, from impaired relaxation to restrictive filling pattern. Cavβ2a TG mouse is a model for HFpEF for exploring HF pathobiology and mechanisms.

Bisphenol S rapidly depresses heart function through estrogen receptor-β and decreases phospholamban phosphorylation in a sex-dependent manner

The health effects of the endocrine disruptor Bisphenol A (BPA) led to its partial replacement with Bisphenol S (BPS) in several products including food containers, toys, and thermal paper receipts. The acute effects of BPS on myocardial contractility are unknown. We perfused mouse hearts from both sexes for 15 min with physiologically relevant doses of BPS or BPA. In females BPS (1 nM) decreased left ventricular systolic pressure by 5 min, whereas BPA (1 nM) effects were delayed to 10 min. BPS effects in male mice were attenuated. In both sexes ER-β antagonism abolished the effects of BPS. Cardiac myofilament function was not impacted by BPS or BPA in either sex, although there were sex-dependent differences in troponin I phosphorylation. BPS increased phospholamban phosphorylation at S16 only in female hearts, whereas BPA reduced phosphorylation in both sexes. BPA decreased phospholamban phosphorylation at T17 in both sexes while BPS caused dephosphorylation only in females. This is the first study to compare sex differences in the acute myocardial response to physiologically relevant levels of BPS and BPA, and demonstrates a rapid ability of both to depress heart function. This study raises concerns about the safety of BPS as a replacement for BPA.

NOX Inhibition Improves β-Adrenergic Stimulated Contractility and Intracellular Calcium Handling in the Aged Rat Heart

Cardiac aging is characterized by alterations in contractility and intracellular calcium ([Ca2+]i) homeostasis. It has been suggested that oxidative stress may be involved in this process. We and others have reported that in cardiomyopathies the NADPH oxidase (NOX)-derived superoxide is increased, with a negative impact on [Ca2+]i and contractility. We tested the hypothesis that in the aged heart, [Ca2+]i handling and contractility are disturbed by NOX-derived superoxide. For this we used adults (≈5 month-old) and aged (20–24 month-old) rats. Contractility was evaluated in isolated hearts, challenged with isoproterenol. To assess [Ca2+]i, isolated cardiac myocytes were field-stimulated and [Ca2+]i was monitored with fura-2. Cardiac concentration-response to isoproterenol was depressed in aged compared to adults hearts (p < 0.005), but was restored by NOX inhibitors apocynin and VAS2870. In isolated cardiomyocytes, apocynin increased the amplitude of [Ca2+]i in aged myocytes (p < 0.05). Time-50 [Ca2+]i decay was increased in aged myocytes (p < 0.05) and reduced towards normal by NOX inhibition. In addition, we found that myofilaments Ca2+ sensitivity was reduced in aged myocytes (p < 0.05), and was further reduced by apocynin. NOX2 expression along with NADPH oxidase activity was increased in aged hearts. Phospholamban phosphorylation (Ser16/Thr17) after isoproterenol treatment was reduced in aged hearts compared to adults and was restored by apocynin treatment (p < 0.05). In conclusion, β-adrenergic-induced contractility was depressed in aged hearts, and NOX inhibition restored back to normal. Moreover, altered Ca2+ handling in aged myocytes was also improved by NOX inhibition. These results suggest a NOX-dependent effect in aged myocytes at the level of Ca2+ handling proteins and myofilaments.