Rationale Induction of the fetal hypertrophic marker gene beta-myosin heavy chain (β-MyHC) is a signature feature of pressure overload hypertrophy in rodents. β-MyHC is assumed present in all or most enlarged myocytes. Objective To quantify the number and size of myocytes expressing endogenous β-MyHC using a flow cytometry approach. Methods and Results Myocytes were isolated from the LV of male C57Bl/6J mice after transverse aortic constriction (TAC), and the fraction of cells expressing endogenous β-MyHC was quantified by flow cytometry on 10,000–20,000 myocytes, using a validated β-MyHC antibody. Side scatter by flow cytometry in the same cells was validated as an index of myocyte size. β-MyHC-positive myocytes were 3±1% of myocytes in control hearts (n=12), increasing to 25±10% at 3d-6w after TAC (n=24, p<0.01). β-MyHC-positive myocytes did not enlarge with TAC, and were smaller at all times than myocytes without β-MyHC (~70% as large, p<0.001). β-MyHC-positive myocytes arose by addition of β-MyHC to α-MyHC, and had more total MyHC after TAC than did the hypertrophied myocytes that had α-MyHC only. Myocytes positive for β-MyHC were found in discrete regions of the LV, in 3 patterns, peri-vascular, in areas with fibrosis, and in apparently normal myocardium. Conclusion β-MyHC protein is induced by pressure overload in a minor sub-population of smaller cardiac myocytes. The hypertrophied myocytes after TAC have α-MyHC only. These data challenge the current paradigm of the fetal hypertrophic gene program, and identify a new sub-population of smaller working ventricular myocytes with more myosin.

Rationale It is unknown if every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), beta-1, beta-2, beta-3, alpha-1A, and alpha-1B. The beta-1 and beta-2 are thought to be the dominant myocyte ARs. Objective Quantify the 5 cardiac ARs in individual ventricular myocytes. Methods and Results We studied ventricular myocytes from wild type mice, mice with alpha-1A and alpha-1B knockin reporters, and beta-1 and beta-2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of ERK and phospholamban), and contraction. We found that the beta-1 and alpha-1B were present in all myocytes. The alpha-1A was present in 60%, with high levels in 20%. The beta-2 and beta-3 were detected in only about 5% of myocytes, mostly in different cells. In intact heart, 30% of total beta-ARs were beta-2 and 20% were beta-3, both mainly in nonmyocytes. Conclusion The dominant ventricular myocyte ARs present in all cells are the beta-1 and alpha-1B. The beta-2 and beta-3 are mostly absent in myocytes but are abundant in nonmyocytes. The alpha-1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with beta-1 and alpha-1B only; 60% that also have the alpha-1A; and 5% each that also have the beta-2 or beta-3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms.

Background Translation of preclinical findings could benefit from a simple, reproducible, high throughput human model to study myocardial signaling. Alpha-1A-adrenergic receptors (ARs) are expressed at very low levels in the human heart, and it is unknown if they function. Objectives To develop a high throughput human myocardial slice culture model, and to test the hypothesis that alpha-1A- ARs are functional in the human heart. Methods Cores of LV free wall 8 mm diameter were taken from 52 hearts (18 failing and 34 nonfailing). Slices 250 μm thick were cut with a Krumdieck apparatus and cultured using a rotating incubation unit. Results About 60 slices were cut from each LV core, and a typical study could use 96 slices. Myocyte morphology was maintained, and diffusion into the slice center was rapid. Slice viability was stable for at least 3 days in culture by ATP and MTT assays. The beta-AR agonist isoproterenol stimulated phospholamban phosphorylation, and the alpha-1A-AR agonist A61603 stimulated ERK phosphorylation, with nanomolar EC50 values in slices from both failing and nonfailing hearts. Strips cut from the slices were used to quantify activation of contraction by isoproterenol, A61603, and phenylephrine. The slices supported transduction by adenovirus. Conclusions We have developed a simple, high throughput LV myocardial slice culture model to study signaling in the human heart. This model can be useful for translational studies, and we show for the first time that the alpha-1A-AR is functional in signaling and contraction in the human heart.

Alpha-1 adrenergic receptors mediate adaptive effects in the heart and cardiac myocytes, and a myocyte survival pathway involving the alpha-1A receptor subtype and ERK activation exists in vitro. However, data in vivo are limited. Here we tested A61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide), a selective imidazoline agonist for the alpha-1A. A61603 was the most potent alpha-1-agonist in activating ERK in neonatal rat ventricular myocytes. A61603 activated ERK in adult mouse ventricular myocytes and protected the cells from death caused by the anthracycline doxorubicin. A low dose of A61603 (10 ng/kg/d) activated ERK in the mouse heart in vivo, but did not change blood pressure. In male mice, concurrent subcutaneous A61603 infusion at 10 ng/kg/d for 7 days after a single intraperitoneal dose of doxorubicin (25 mg/kg) increased survival, improved cardiac function, heart rate, and cardiac output by echocardiography, and reduced cardiac cell necrosis and apoptosis and myocardial fibrosis. All protective effects were lost in alpha-1A-knockout mice. In female mice, doxorubicin at doses higher than in males (35–40 mg/kg) caused less cardiac toxicity than in males. We conclude that the alpha-1A-selective agonist A61603, via the alpha-1A adrenergic receptor, prevents doxorubicin cardiomyopathy in male mice, supporting the theory that alpha-1A adrenergic receptor agonists have potential as novel heart failure therapies.

Alpha-1-adrenergic receptors (α1-ARs) regulate coronary arterial blood flow by binding catecholamines, norepinephrine (NE), and epinephrine (EPI), causing vasoconstriction when the endothelium is disrupted. Among the three α1-AR subtypes (α1A, α1B, and α1D), the α1D subtype predominates in human epicardial coronary arteries and is functional in human coronary smooth muscle cells (SMCs). However, the presence or function of α1-ARs on human coronary endothelial cells (ECs) is unknown. Here we tested the hypothesis that human epicardial coronary ECs express functional α1-ARs. Cultured human epicardial coronary artery ECs were studied using quantitative real-time reverse transcription polymerase chain reaction, radioligand binding, immunoblot, and 3H-thymidine incorporation. The α1B-subtype messenger ribonucleic acid (mRNA) was predominant in cultured human epicardial coronary ECs (90–95% of total α1-AR mRNA), and total α1-AR binding density in ECs was twice that in coronary SMCs. Functionally, NE and EPI through the α1B subtype activated extracellular signal-regulated kinase (ERK) in ECs, stimulated phosphorylation of EC endothelial nitric oxide synthase (eNOS), and increased deoxyribonucleic acid (DNA) synthesis. These results are the first to demonstrate α1-ARs on human coronary ECs and indicate that the α1B subtype is predominant. Our findings provide another potential mechanism for adverse cardiac effects of drug antagonists that nonselectively inhibit all three α1-AR subtypes.

Although major metabolites of some antidepressant drugs are know to be active, their pharmacological effects are poorly characterized. Two of the most selective antidepressants, desipramine (selectively inhibits norepinephrine reuptake) and citalopram (selectively inhibits serotonin reuptake) are frequently used in animal studies of antidepressant action, as well as being useful therapeutically. The primary aim of this study was to determine the affinity of desmethyldesipramine, an active metabolite of desipramine, for the rat norepinephrine and serotonin transporters, as well as for the rat α 2 -adrenoceptor. The pharmacological characteristics of desmethyldesipramine and desmethylcitalopram, an active metabolite of citalopram, were also determined for various human transporters and neurotransmitter receptors. Competition binding studies using [³H]nisoxetine and [³H]citalopram showed desipramine to be 25 times more selective for the rat norepinephrine as compared to serotonin transporter (6.2 nM vs. 158 nM) whereas desmethyldesipramine is 12 times more selective for the serotonin over the norepinephrine transporter (12.8 nM vs. 153 nM). Interestingly, the affinity of desmethyldesipramine for the serotonin transporter is similar to the affinity of desipramine for the norepinephrine transporter. Desipramine and desmethyldesipramine were found to have a lower affinity for the rat α 2A(D) -adrenoceptor than the transporters, suggesting that this receptor is not a major site of action for either compound. Thus, the pharmacological effects of desipramine in rats may be attributed not only to the inhibition of the norepinephrine transporter by desipramine but also to the inhibition of serotonin transporter by the active metabolite desmethyldesipramine.

Introduction Alpha-1-adrenergic receptors (α1-ARs) are G-protein coupled receptors (GPCRs) with three highly homologous subtypes (α1A, α1B, and α1D). Of these three subtypes, only the α1A and α1B are expressed in the heart. Multiple pre-clinical models of heart injury demonstrate cardioprotective roles for the α1A. Non-selective α1-AR activation promotes glycolysis in the heart, but the functional α1-AR subtype and broader metabolic effects have not been studied. Objectives Given the high metabolic demands of the heart and previous evidence indicating benefit from α1A activation, we chose to investigate the effects of α1A activation on the cardiac metabolome in vivo. Methods Mice were treated for one week with a low, subpressor dose of A61603, a highly selective and potent α1A agonist. Cardiac tissue and serum were analyzed using a non-targeted metabolomics approach. Results We identified previously unrecognized metabolic responses to α1A activation, most notably broad reduction in the abundance of polyunsaturated fatty acids (PUFAs) and endocannabinoids (ECs). Conclusion Given the well characterized roles of PUFAs and ECs in inflammatory pathways, these findings suggest a possible role for cardiac α1A-ARs in the regulation of inflammation and may offer novel insight into the mechanisms underlying the cardioprotective benefit of selective pharmacologic α1A activation.

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