Abstract
In an integrative approach, we studied cardiac effects of recently published novel H2 receptor agonists in the heart of mice that overexpress the human H2 receptor (H2-TG mice) and littermate wild type (WT) control mice and in isolated electrically driven muscle preparations from patients undergoing cardiac surgery. Under our experimental conditions, the H2 receptor agonists UR-Po563, UR-MB-158, and UR-MB-159 increased force of contraction in left atrium from H2-TG mice with pEC50 values of 8.27, 9.38, and 8.28, respectively, but not in WT mice. Likewise, UR-Po563, UR-MB-158, and UR-MB-159 increased the beating rate in right atrium from H2-TG mice with pEC50 values of 9.01, 9.24, and 7.91, respectively, but not from WT mice. These effects could be antagonized by famotidine, a H2 receptor antagonist. UR-Po563 (1 µM) increased force of contraction in Langendorff-perfused hearts from H2-TG but not WT mice. Similarly, UR-Po563, UR-MB-158, or UR-MB-159 increased the left ventricular ejection fraction in echocardiography of H2-TG mice. Finally, UR-Po563 increased force of contraction in isolated human right atrial muscle strips. The contractile effects of UR-Po563 in H2-TG mice were accompanied by an increase in the phosphorylation state of phospholamban. In summary, we report here three recently developed agonists functionally stimulating human cardiac H2 receptors in vitro and in vivo. We speculate that these compounds might be of some merit to treat neurologic disorders if their cardiac effects are blocked by concomitantly applied receptor antagonists that cannot pass through the blood-brain barrier or might be useful to treat congestive heart failure in patients.
SIGNIFICANCE STATEMENT Recently, a new generation of histamine H2 receptor (H2R) agonists has been developed as possible treatment option for Alzheimer’s disease. Here, possible cardiac (side) effects of these novel H2R agonists have been evaluated.
Introduction
Histamine is a mediator of many physiologic processes like inflammation, allergy, gastric acid secretion, hematopoiesis, cell proliferation, and wound healing (Parsons and Ganellin, 2006). In the central nervous system, histamine is involved in awakening and sleep, regulation of body weight, learning, perception of pain, and memory (Jørgensen et al., 2007). Moreover, histamine dysfunction is probably involved in diseases like narcolepsy, depression, and Alzheimer’s disease (Mehta et al., 2021). If one wants to treat, for instance, Alzheimer’s disease in the central nervous system with a peroral H2 receptor (H2R) agonist, one also treats the patient’s heart. In the heart, histaminergic cardiac effects were initially described by Dale and Laidlaw (1910) and Ackermann and Kutscher (1910). In the human heart, H1 and H2 receptors have been identified using antibodies (Matsuda et al., 2004). There are many more H1 receptors in human atrial samples than H2 receptors (Baumann et al., 1983; Matsuda et al., 2004). Nevertheless, the H2 receptors currently thought to be responsible for the histaminergic positive inotropic and positive chronotropic effects in the human atrium (Levi et al., 1981; Sanders et al., 1996). These effects are elicited by H2 receptor agonists and not H1 receptor agonists because the positive inotropic effects of histamine are blocked by H2 receptor antagonists like cimetidine and famotidine (Seifert et al., 2013; Panula et al., 2015). In human atrial samples, the stimulation of H2 receptors led to an increase in cAMP content, an elevated activity of the cAMP dependent protein kinase (Sanders et al., 1996) (scheme in Fig. 1), and subsequently to an increase in the phosphorylation state of phospholamban (PLB) (scheme in Fig. 1) and the inhibitory subunit of troponin (Neumann et al., 2021b). In isolated human atrial or ventricular preparations, besides histamine, other histamine receptor agonists like amthamine (pD2 = 5.38) or the more potent agonist impromidine (pD2 = 6.59) have been studied (Bristow et al., 1982; Poli et al., 1994; Coruzzi et al., 1995). More recently, we have generated a transgenic mouse model that overexpresses the human H2 receptor in the murine heart (Gergs et al., 2019). In the hearts of this model (H2-TG mice) but not in the wild type (WT) littermates, we could identify the human H2 receptor mRNA and the protein by immunohistology of ventricular slices as well as by autoradiography of hearts with a radioactively labeled H2R agonist but not in Western blotting experiments (Gergs et al., 2019; Gergs et al., 2020; Neumann et al., 2021b). Moreover, contractile effects to histamine were only present in living H2-TG mice, isolated perfused hearts from H2-TG mice, isolated cells from H2-TG mice, and isolated atrial preparations from H2-TG mice, but not those from WT mice (Gergs et al., 2019; Gergs et al., 2020; Neumann et al., 2021a; Neumann et al., 2021b). Therefore, we had suggested that this H2-TG model might be a useful model to study human H2R in all regions of the heart. It is important to point out that we used an alpha myosin heavy chain promoter for the human H2R transgene, meaning that we overexpressed the human H2R only in the cardiomyocytes of the heart (Gergs et al., 2019).
There are studies reporting that stimulation of postsynaptic H2Rs in the brain might be beneficial for learning and memory and could therefore be interesting in the treatment of, e.g., Alzheimer’s disease (Khan et al., 2016). As these effects have only been shown with dual-acting acetylcholinesterase inhibitors and H3R antagonists initiating this process by inhibiting presynaptic H3-autoreceptors (Darras et al., 2014; Khan et al., 2016; Sadek et al., 2016) the use of central nervous system–penetrating H2R agonists is of great interest. In the present work we wanted to test whether the recently published H2R agonists UR-Po563 (Biselli et al., 2021), UR-MB-158, and UR-MB-159 (Tropmann et al., 2021) (Fig. 1) act on the human cardiac H2R.
Materials and Methods
Transgenic Mice
The investigation conforms to the Guide for the Care and Use of Laboratory Animals (National Research Council Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 2011). Animals were maintained and handled according to approved protocols of the animal welfare committee of the University of Halle-Wittenberg, Germany.
The plasmid, containing the human H2 receptor cDNA (GenBank accession number AY136744), was kindly provided by R. Seifert (Institute of Pharmacology, Hannover Medical School, Hannover, Germany). Generation of the transgenic mice with cardiomyocyte-specific expression of the human H2R was performed by the TRAM unit of the Westfälische Wilhelms-Universität, Münster, Germany, and has been described before (Gergs et al., 2019). Genotypes were identified by polymerase chain reaction analyses of tail tip DNA using the following primers: 5′-ACCCTTACCCCACATAGACC-3′ and 5′-AGCAGGTCAGTGATAGCCAA-3′. The polymerase chain reaction was performed using the Ampliqon Taq DNA polymerase (Biomol, Hamburg, Germany) according to the manufacturer’s instructions. For the experiments, 3–6-month-old H2-TG mice and WT littermates (age-matched) of both sexes (evenly distributed) were used.
Western Blot Analysis
Homogenates from ventricular tissue samples were prepared in 300 µl of 10 mM NaHCO3 and 100 µl 20% SDS. Crude extracts were incubated at 25°C for 30 minutes before centrifugation to remove debris, and thereafter the supernatants (= homogenates) were separated and stored at −80°C until further use. Western blot analysis was performed as previously described (Gergs et al., 2004). Briefly, aliquots of 100 µg of protein were loaded per lane, and bands were detected using enhanced chemifluorescence (GE Healthcare Europe, Freiburg, Germany) together with a Typhoon 9410 Variable Mode Imager (GE Healthcare Europe, Freiburg, Germany). The following primary antibodies were used in this study: polyclonal rabbit anti-calsequestrin (SP5340P, Acris Antibodies, Herford, Germany) and polyclonal rabbit anti–phospho-PLB [antibodies were raised against PLB-peptide phosphorylated at serine 16 (PS16-PLB), A010-12, Badrilla, Leeds, UK]. The characteristics and use of these antibodies have been reported repeatedly by our group (Kirchhefer et al., 2002). The specificity of the anti-calsequestrin antibody was controlled by running in parallel cardiac samples from mice with calsequestrin deletion. In these knockout mice the antibody detected no signal at 58 kDa, the size of calsequestrin (Gergs et al., 2017). Specificity of anti-phosphorylated PLB antibodies was ascertained by running next to each other boiled and unboiled samples from hearts treated with isoproterenol (to increase PLB phosphorylation). Boiling reduces the apparent molecular weight of PLB from about 27 kDa (pentameric form) to values around 10 kDa (monomeric form) under these experimental conditions (Neumann et al., 2021a).
Echocardiography
Echocardiography in spontaneously breathing mice was performed under anesthesia with 1.5% isoflurane (Gergs et al., 2010). We injected the dihydrochloride salts of UR-Po563, UR-MB-158, or UR-MB-159 (dissolved in water) or famotidine as 100 µl of a 1 mM stock solution into the peritoneum of H2-TG or WT mice. First famotidine was injected, and five minutes later UR-Po563, UR-MB-158, or UR-MB-159. This was done to offer enough time for the H2R antagonist famotidine to occupy the cardiac H2R. After five additional minutes the left ventricle was assessed using B-mode to obtain an overall view. The recording was then changed to M-mode to quantify the function of the left ventricle by measuring the ejection fraction of the left ventricle using the software supplied by the manufacturer (Vevo 2100, Visual Sonic, Toronto, Canada).
Contractile Function
Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg kg−1), and hearts were excised. Right and left atria were dissected from isolated H2-TG and WT mice hearts and mounted in an organ bath. Left atrial preparations were continuously electrically stimulated (field stimulation) with each impulse consisting of 1 Hz, with a voltage of 10–15% above threshold and 5 ms duration. Right atrial preparations were allowed to contract spontaneously. The bathing solution contained (in mM) NaCl 119.8, KCI 5.4, CaCl2 1.8, MgCl2 1.05, NaH2P04 0.42, NaHCO3 22.6, Na2EDTA 0.05, ascorbic acid 0.28, and glucose 5.0, continuously gassed with 95% O2 and 5% CO2 and maintained at 35°C resulting in a pH of 7.4. Signals detected via an isometric force transducer were amplified and continuously recorded. UR-Po563, UR-MB-158, or UR-MB-159 was cumulatively applied to the organ bath. After three changes of the buffer in the organ bath of the indicated experiments, famotidine was applied (1 µM in the organ bath), and then cumulative addition of UR-Po563, UR-MB-158, or UR-MB-159 to the organ bath was repeated. This was done to assess the ability of famotidine to antagonize the contractile effects of the respective agonist in the organ bath.
Langendorff-Perfused Hearts
Heart preparations were used as described previously (Kirchhefer et al., 2014). Mice were anesthetized intraperitoneally with pentobarbital sodium (50 mg kg−1) and treated with 1.5 units of heparin. The hearts were removed from the opened chest, immediately attached by the aorta to a 20-gauge cannula, and perfused retrogradely under constant flow of 2 ml min−1 with oxygenized buffer solution (37°C) containing (in mM) NaCl 119.8, KCI 5.4, CaCl2 1.8, MgCl2 1.05, NaH2P04 0.42, NaHCO3 22.6, Na2EDTA 0.05, ascorbic acid 0.28, and glucose 5.0 in an isolated heart system. The heart preparations were allowed to equilibrate for 30 minutes before measurements. The developed force was measured with a hook applied to the apex cordis that was connected via a return pulley to a force transducer. The data were recorded using a PowerLab system (ADInstruments, Oxford, UK). Ventricular contractions were measured and monitored continuously. The beating rate and the first derivative of the developed force (+dF/dt and −dF/dt) were calculated electronically using the chart software (ADInstruments, Oxford, UK).
Human Atrium
Human atrial preparations were used as previously described (Neumann et al., 2021a). Right atrial samples were obtained from patients who underwent cardiac bypass surgery because of a three-vessel coronary artery disease. From these right atrial samples, we cut small trabeculae carneae and handled them exactly like mouse atrial samples. In brief, they were mounted in organ baths, attached to an isometric force transducer, and stimulated electrically at 1 Hz, and the buffer had the same composition as described above. The samples were freeze clamped in liquid nitrogen after the experiment to stop all biochemical reactions and to maintain the phosphorylation state of the proteins of interest. From some atrial samples, small cardiac strips were prepared and incubated in 1.5 ml reaction tubes, drugs were added, and then cardiac strips were rapidly frozen. These procedures have been reported in detail before (Gergs et al., 2009; Neumann et al., 2021c). Here, five atrial preparations from three patients could be used. The patient characteristics are described as follows: age, 52–72 years old; sex, male; New York Heart Association class, III–IV; Canadian Cardiovascular Society angina grading scale, III; left ventricular ejection fraction, 40–60%; further diagnoses were arterial hypertension, hypercholesterolemia, and diabetes (two of three); and medications included anticoagulants, platelet aggregation inhibitors, β-adrenoceptor antagonists, calcium channel blockers (dihydropyridines), angiotensin converting enzyme inhibitors or angiotensin receptor blockers, diuretics, proton-pump inhibitors, metformin, or insulin. The study complied with the Declaration of Helsinki and was approved by the Ethics Committee of the University of Halle-Wittenberg (hm-bü 04.08.2005). All patients gave informed consent.
Mini-G Protein Recruitment Assay
The mini-G protein recruitment assay was performed as previously described using HEK293T cells stably expressing NlucN-mGs/hH2R-NlucC or NlucN-mGs/gpH2R-NlucC and UR-Po563 in various concentrations (Höring et al., 2020; Tropmann et al., 2021).
Data Analysis
Data shown are means ± S.E.M. Statistical significance was estimated by a paired or unpaired Student’s t test for the concentration response curves or Langendorff experiments or by a two-way analysis of variance (ANOVA) followed by Bonferroni’s posttest for the echocardiographic and Western blot results. A value of P < 0.05 was considered significant.
Drugs and Materials
UR-Po563 (Biselli et al., 2021), UR-MB-158, and UR-MB-159 (Tropmann et al., 2021) were synthesized as dihydrochloride salts according to the literature (Fig. 1). All other chemicals were of analytical grade. Demineralized water was used throughout the experiments. Stock solutions were freshly prepared daily.
Results
In isolated electrically (1 Hz) driven left atrial preparations, histamine, cumulatively applied, increased force of contraction in preparations from H2-TG mice (original recording in Fig. 2A) but failed to affect force of contraction in preparations from WT mice (Fig. 2B). The data are summarized in Fig. 2, B–E. Moreover, histamine concentration dependently increased the velocity of contraction (maximum rate of tension development: Fig. 2C) and decreased the velocity of relaxation (minimum rate of tension development: Fig. 2D). Similarly, in spontaneously contracting right atrial preparations from WT mice, histamine, cumulatively given, did not increase the beating rate. However, in H2-TG right atria, histamine augmented the beating rate in a time- and concentration-dependent fashion (Fig. 2E). These findings are in line with our previous reports (Gergs et al., 2019; Gergs et al., 2020; Neumann et al., 2021a; Neumann et al., 2021b). The effects of histamine in H2-TG on force of contraction and beating rate were antagonized by H2R antagonists applied 30 minutes before application of histamine as shown in previous reports where we used either cimetidine (Gergs et al., 2019) or famotidine (Neumann et al., 2021a) to antagonize the histamine effects.
Similar to histamine (Fig. 2), we noted that UR-Po563 increased force of contraction in a time- and concentration-dependent manner in atrial preparations only from H2-TG. This and the antagonistic effect of famotidine can be seen in the original recordings in Fig. 3A. The data are summarized in Fig. 3, B–E. The respective EC50 values of UR-Po563 are provided in Supplemental Table 1 and can be compared with values from UR-MB-158, UR-MB-159, histamine, and those of some typical older histamine H2R ligands. UR-Po563 increased the beating rate in preparations from H2-TG but not WT mice (Fig. 3C). In the left atrial samples, UR-Po563 concentration- and time-dependently increased the first derivative of force versus time dF/dt in absolute terms (Fig. 3, D and E). The effects of UR-Po563 on force of contraction, its first derivative, or the beating rate in H2-TG were antagonized by famotidine (Fig. 3).
In a more potent way (Supplemental Table 1), UR-MB-158 increased the force of contraction and the beating rate in atrial preparations from H2-TG but not WT mice in a concentration- and time-dependent manner. This can be seen in an original recording (Fig. 4A) and is summarized in Fig. 4, B–E. UR-MB-158 increased the beating rate in right atrial preparations from H2-TG but not WT mice (Fig. 4C). Moreover, in the left atrial samples, UR-MB-158 increased the first derivative of force versus time dF/dt in absolute terms (Fig. 4, D and E). The effects of UR-MB-158 on force of contraction or beating rate were antagonized by famotidine (Fig. 4).
UR-MB-159 was also more potent than histamine under the present experimental conditions (Supplemental Table 1): UR-MB-159 increased force of contraction (original tracing: Fig. 5A and summary: Fig. 5B) and beating rate (Fig. 5C) in a time- and concentration-dependent manner in atrial preparations from H2-TG but not WT mice. In the left atrial samples, UR-MB-159 increased the maximum and minimum first derivative of force versus time (dF/dt, Fig. 5, D and E). The effects of UR-MB-159 in left and right atria were antagonized by famotidine (Fig. 5).
Next, it was of interest to investigate ventricular effects of histamine. To that end, we used isolated retrogradely perfused hearts (Langendorff preparations). These preparations were allowed to beat spontaneously. We recorded force of contraction from the apex; therefore, we measured left ventricular force under these conditions. We noted that 1 µM of UR-Po563 increased force of contraction in hearts from H2-TG but not WT mice (Table 1). This was in line with our previous reports on histamine in Langendorff-perfused hearts (Gergs et al., 2019; Gergs et al., 2021; Neumann et al., 2021a; Neumann et al., 2021b).
To investigate the effects of the novel H2 receptor agonists on cardiovascular performance in vivo, we performed echocardiographic measurements in H2-TG and WT mice under anesthesia (original M-mode recordings: Fig. 6A). We noted that intraperitoneally injected UR-Po563 (100 µl of a 1 mM solution) increased left ventricular ejection fraction and beating rate in H2-TG mice (Table 2; Fig. 6B). Furthermore, using echocardiography, we studied typical dimensions of the heart like left ventricular systolic and diastolic diameters under basal and UR-Po563-stimulated conditions. We noted substantial alterations in these parameters after injection of UR-Po563 in H2-TG but not in WT mice (Fig. 6). These results are also reminiscent of our previous studies with histamine itself under these conditions (Gergs et al., 2019). Effects of UR-Po563 were antagonized by famotidine (Table 2; Fig. 6B). Interestingly, we had the opportunity to study atrial preparations of three animals in which UR-Po563 was injected and from which the heart could be harvested (under general anesthesia) after performing echocardiography. To our surprise in left atrial preparations from H2-TG, where UR-Po563 exerted a profound increase in ejection fraction, UR-Po563 was unable to increase force of contraction under the conditions described in Fig. 2 (data not shown), despite the fact that we exchanged the organ bath buffer (10 ml volume) three times before we started the contraction experiment. Thus, one can speculate that UR-Po563 sticks quite tight to cardiac tissue, but the exact reasons remain to be elucidated.
Furthermore, to better understand the underlying signal transduction mechanism (Fig. 1), we assessed the phosphorylation state of PLB in atrial and ventricular preparations by Western blotting of the frozen samples with a phosphorylation state sensitive antibody. As expected from our previous reports with histamine (Gergs et al., 2019; Gergs et al., 2021; Neumann et al., 2021a; Neumann et al., 2021b), UR-Po563 increased the phosphorylation of PLB at serine 16 in left and right atrial preparations from H2-TG mice (freeze clamped at the maximum of the positive inotropic effect) (Fig. 7). In contrast, in atrial preparations from WT mice, UR-Po563 failed to increase the phosphorylation state of PLB (Fig. 7). The data are summarized as scatter plots. Similar results were obtained in freeze clamped isolated perfused heart: UR-Po563 (1 µM) increased the phosphorylation state of PLB in preparations from H2-TG [freeze clamped at the maximum of the positive inotropic effect (PIE)] but not WT mice (Fig. 8).
Finally, it was of interest whether UR-Po563, like histamine itself, could increase force of contraction in the human heart. As a model we used electrically driven (1 Hz) isolated right atrial strips obtained from the operating theater in routine bypass surgery. Here, we noted that UR-Po563 concentration-dependently could increase force of contraction in atrial preparations of human hearts (Fig. 9A: original tracings; Fig. 9, B–E: summary) and increased dF/dt (Fig. 9, D and E).
Discussion
In our integrative approach, we have functionally shown that UR-Po563, UR-MB-158 and UR-MB-159 can stimulate human cardiac H2 receptors. The substances described have already been characterized in two detailed studies (Biselli et al., 2021; Tropmann et al., 2021) with regard to receptor binding (Supplemental Table 2) and functionality (G protein recruitment, β-arrestin2 recruitment, and guinea pig right atrium; Supplemental Tables 3 and 4). The published data were supplemented in this study for UR-Po563 (Supplemental Tables 3 and 4) and summarized in Supplemental Tables 2, 3 and 4 for a better comparability. In addition to the detailed characterization of H2R orthologs, statements on selectivity within the family of histamine receptors can also be found in Supplemental Table 2.
Previously, dimaprit has been developed as a H2R agonist, devoid of action at H1 receptors (Panula et al., 2015). However, later (when H3 and H4 receptors were identified, cloned, and characterized), dimaprit was found also to be an agonist at H3 and H4 receptors. Amthamine was the next step to find a selective agonist at the H2R (Panula et al., 2015). In some systems, histamine and dimaprit were equipotent [U937 cells (Smit et al., 1994)]; in other cells, histamine was more potent than dimaprit [H2-transfected CHO cells (Smit et al., 1994)]. In the human heart, dimaprit was a full agonist for its PIE with an EC50 value of 43 µM (Poli et al., 1994). However, in other laboratories, dimaprit was more potent for its PIE (human left atrium, right atrium, papillary muscle) with an EC50 around 3 µM (Bristow et al., 1982; Eckel et al., 1982; Brown et al., 1986). In the guinea pig papillary muscle, amthamine was more potent and equieffective to histamine with respect to the PIE (Poli et al., 1993). Amthamine, compared with histamine, acts as a full agonist with respect to PIE in human right atrial preparations with an EC50 value of 4.2 µM (Poli et al., 1994). In contrast, in the guinea pig right atrium, amthamine was more potent than histamine with respect to the positive chronotropic effect (Poli et al., 1993). Interestingly, histamine was more potent in the H2-TG mouse atrium with respect to the PIE than in human atrium, although the receptor was the same (Supplemental Table 1). This clearly demonstrates the limitation of model systems (animal models or cell cultures). Species differences and differences in, e.g., receptor density must always be considered. Unfortunately, it was not possible to quantify the expression level of the H2R in the H2-TG mice by either Western blotting or radioligand binding in heart homogenates or membrane preparations (Gergs et al., 2019). In this context, it is not surprising that UR-Po563 is more potent in H2-TG atrial preparations compared with human atrial preparations.
PLB is only expressed in cardiomyocytes and not in noncardiomyocytes. The fact that we measured an increase in PLB phosphorylation in atrial and ventricular preparations is strongly indicative that UR-Po563 acts on H2 receptors in cardiomyocytes.
The fact that we measured an increase in serine 16 phosphorylation of PLB is easily interpreted as a result from cAMP generation after UR-Po563 application in H2-TG mice. H2R activation is expected to activate protein kinase A (Fig. 1), which only phosphorylates PLB on serine 16 and which is thought to augment cardiac relaxation (Simmerman et al., 1986). More recently, we have shown that H2R stimulation leads to PLB phosphorylation in isolated atrial preparations from H2-TG but not WT mice (Gergs et al., 2019). In addition, we could measure that histamine increases the phosphorylation in the human heart, more specifically in isolated electrically stimulated (1 Hz) right atrial preparations from patients undergoing heart surgery (Neumann et al., 2021a).
Clinical Relevance
In contrast to β1 adrenoceptors, the density of H2 receptors in the failing human hearts were not changed compared with nonfailing controls (ventricular samples from transplants in Munich, Germany); Baumann et al. (1984) used tritiated tiotidine for binding studies). In isolated ventricular preparations from failing human hearts, Bauman et al. did not detect a reduced PIE to histamine compared with nonfailing controls (Baumann et al., 1982; Baumann et al., 1983; Baumann et al., 1984) and suggested that H2R agonists might be clinical useful for the treatment of heart failure. They even tested a H2R agonist (impromidine) in the intensive care unit in patients and described beneficial acute effects but also serious side effects like increased gastric acid (Baumann et al., 1984; Felix et al., 1995). In contrast, others noted a reduced PIE to histamine in samples from failing human hearts (Brown et al., 1986; Böhm et al., 1988).
There are interesting data that H2 blockers reduce the propensity for heart failure in humans. Apparently, the first data in this regard were Japanese register data (Kim et al., 2004); this prompted a better controlled but still retrospective study in the United States. Leary et al. (2014, 2016b) provided the first evidence for a beneficial role of H2R blockers by delaying the onset of heart failure and alterations of the morphology of the heart: the development of left ventricular dilation, a hallmark of imminent heart failure, was delayed. They suggested furthermore that this might be an example of repurposing of drugs (antihistamines) for heart failure (Leary et al., 2016a; Leary and Bristow, 2016). In a subsequent analysis, they suggested that it might be possible to identify a subgroup of patients with risk of heart failure, which might benefit particularly from treatment with H2R antagonists (Leary et al., 2018).
Hence, one could argue for a twofold clinical utility of the present data. If stimulation of H2 receptors is a valid concept to increase force of contraction in patients, UR-MB-158 and UR-MB-159 are more potent than histamine and older H2R agonists in functional studies on human H2 receptors. Thus, they may be tested for patients with end stage heart failure to sustain contractility under clinical conditions. Alternatively, if it turns out that H2R agonists are useful for the treatment of patients with Alzheimer’s disease, it might be conceivable that drugs like UR-Po563, UR-MB-158, or UR-MB-159 might be tested. Here, the cardiac effects might be unwanted side effects, and one might block these peripheral effects by treating the patients concomitantly with a H2R antagonist that does not pass through the blood-brain barrier. That was why we used famotidine in this experimental study. It is known that famotidine in contrast to cimetidine does not pass the human blood-brain barrier. On the other hand, the preliminary experiments with human atrial preparations presented here indicate a relatively low potency of the new H2R agonist UR-Po563 with respect to the PIE in the human atrium. Therefore, the therapeutic dose necessary to treat Alzheimer’s disease may be at least in part safe with respect to cardiac side effects. However, the cardiac response to H2R agonists may vary in a wide range from patient to patient, in contrast to a genetically homogenous mouse model. Moreover, patients differ widely in their diseases and medications, which make comparisons very difficult. Therefore, these aspects have to be studied in more detail in future work. Studies are ongoing in our groups to measure the concentrations of applied UR-Po563, UR-MB-158, and UR-MB-159 in the heart and brain in mice. Moreover, we have started to measure famotidine concentrations in heart and brain of mice. These data might support or repudiate our suggestions.
In summary, we describe that three novel H2R agonists named UR-Po563, UR-MB-158, and UR-MB-159 behave as functional full agonists on human cardiac H2 receptors. They are functionally more potent than histamine in the heart at H2 receptors. They stimulate H2 receptors in the right and left atrium, the ventricle in vitro, and the ventricle in vivo of H2-TG but not WT mice. Finally, UR-Po563 increase force of contraction in the human heart in vitro. These novel agents could now be clinically tested to treat heart failure or Alzheimer's disease.
Acknowledgments
The authors thank Sonja Reber and Pia Willmy for expert technical assistance.
Authorship Contributions
Participated in research design: Gergs, Neumann.
Conducted experiments: Büxel, Bresinsky, Kirchhefer, Fehse, Höring, Hofmann, Marušáková, Čináková, Schwarz, Pockes.
Contributed new reagents or analytic tools: Bresinsky, Höring, Pockes.
Performed data analysis: Büxel, Bresinsky, Fehse, Höring, Marušáková, Čináková, Schwarz, Pockes.
Wrote or contributed to the writing of the manuscript: Gergs, Pockes, Neumann.
Footnotes
- Received July 2, 2021.
- Accepted September 14, 2021.
↵This article has supplemental material available at jpet.aspetjournals.org.
Abbreviations
- H2R
- histamine H2 receptor
- H2-TG
- transgenic and overexpressing human H2R
- pD2
- negative logarithm of the EC50 that is the molar concentration of an agonist that produces 50% of the maximal possible effect
- PIE
- positive inotropic effect
- PLB
- phospholamban
- PS16-PLB
- phospholamban phosphorylated at serine 16
- WT
- wild type
- Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics