Trypanosoma cruzi Infection and Endothelin-1 Cooperatively Activate Pathogenic Inflammatory Pathways in Cardiomyocytes

Trypanosoma cruzi, the causative agent of Chagas'' disease, induces multiple responses in the heart, a critical organ of infection and pathology in the host. Among diverse factors, eicosanoids and the vasoactive peptide endothelin-1 (ET-1) have been implicated in the pathogenesis of chronic chagasic cardiomyopathy. In the present study, we found that T. cruzi infection in mice induces myocardial gene expression of cyclooxygenase-2 (Cox2) and thromboxane synthase (Tbxas1) as well as endothelin-1 (Edn1) and atrial natriuretic peptide (Nppa). T. cruzi infection and ET-1 cooperatively activated the Ca²⁺/calcineurin (Cn)/nuclear factor of activated T cells (NFAT) signaling pathway in atrial myocytes, leading to COX-2 protein expression and increased eicosanoid (prostaglandins E₂ and F_2α, thromboxane A₂) release. Moreover, T. cruzi infection of ET-1-stimulated cardiomyocytes resulted in significantly enhanced production of atrial natriuretic peptide (ANP), a prognostic marker for impairment in cardiac function of chagasic patients. Our findings support an important role for the Ca²⁺/Cn/NFAT cascade in T. cruzi-mediated myocardial production of inflammatory mediators and may help define novel therapeutic targets.     

肥厚预刺激对苯肾上腺素诱导的心肌细胞肥大的保护作用

目的:研究不同肥厚预刺激对苯肾上腺素(Phenylephrine,PE)诱导的心肌细胞肥大的影响。方法:胶原酶联合差速贴壁法分离培养原代SD乳鼠心肌细胞后分组:(1)对照组(常规培养48 h);(2)PE组(50μM PE刺激48 h);(3)不同预刺激+PE组:A,不同浓度的PE(10、20、50μM)预刺激(12 h干预,12 h常规培养);B,PE(50μM)预刺激不同时间(period-1,6 h干预,6 h常规培养;period-2,6 h干预,6 h常规培养,再次6 h干预,6 h常规培养;period-3,8 h干预,8 h常规培养;period-4,12 h干预,12 h常规培养)。预刺激后再用PE(50μM)刺激48 h。经细胞骨架蛋白(α-actining)免疫荧光染色,利用激光共聚焦显微镜观察细胞表型,Image J软件计算心肌细胞表面积,利用实时定量PCR检测肥厚相关标志物表达水平。结果:分离的心肌细胞纯度在90%以上。PE组较对照组心肌细胞明显肥大,细胞表面积增加2.3倍,心肌肥厚标记基因心钠肽(atrial natriuretic peptide,ANP)、脑钠肽(brain natriuretic peptide,BNP)和β肌球蛋白重链(βmyosin heavy chain,βMHC)表达明显升高(P0.05);而不同预刺激+PE组心肌细胞肥大表型明显缓解,其中PE(50μM)两次6 h预刺激最为显著(P0.05)。结论:肥厚预刺激可以减轻PE诱导的心肌细胞肥大的程度,从而对心肌肥厚有保护作用。     

TNF-a在乳鼠心肌细胞中诱导HSP70蛋白表达的研究

热休克蛋白70 (HSP70) 在细胞修复、存活和维持细胞正常功能方面有着重要作用。作为分子伴侣,它起着心肌保护的作用。已经对重症心脏病人的心肌组织进行了蛋白组学研究,得到了HSP70在心衰病人心肌组织中较正常人心肌组织表达升高的结论,并且在血液中得到了进一步的验证。在进一步的离体细胞实验中用不同剂量的肿瘤坏死因子-alpha (TNF-α) 刺激乳鼠心肌细胞,以观察不同时间点HSP70的动态表达情况。培养乳鼠心肌细胞,分别对细胞进行热休克（42 ℃）、TNF-α和缺血缺氧处理,在不同的时间点收获细胞,以观察HSP70的动态表达情况。用免疫化学、ELISA以及Western blotting的方法对HSP70蛋白进行分析。结果表明,在正常对照细胞中基本没有阳性信号出现,而在经缺血缺氧、热休克（42 ℃）以及TNF-α处理的细胞中有明显的阳性表达。以上研究首次在乳鼠心肌细胞中证明TNF-α诱导的HSP70表达具有时间和浓度依赖性。通过运用TNF-α对HSP70蛋白表达影响的研究,初步推断HSP70的表达模式,为体内诱导产生HSP70从而发挥心肌保护作用的研究提供一定的理论基础。     

Interferon-Gamma Promotes Infection of Astrocytes by Trypanosoma cruzi

The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ⁺ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD.     

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for α-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.     

Acute and Chronic Experimental Trypanosoma cruzi Infection in the Rat. Response to Systemic Treatment with Recombinant Rat Interferon-Gamma

We examined the effects of recombinant rat inteferon-gamma (IFN-γ) injections on the parasitologic, serologic, immunologic and histopathologic features of acute and chronic experimental Trypanosoma cruzi (T. cruzi) infections in “l” rats. Upon infection at weaning, two rat groups were allocated to receive a 20-day cycle of IFN-γ injections, 20,000 IU/rat each, which started at 1, and 7 days post-infection (pi). Treatment with IFN-γ, initiated at either 1 or 7 days pi, resulted in comparatively lower peak parasitemias (P<0.02) but in similar levels of anti-T. cruzi circulating antibodies and serum IFN-γ activities. The latter appeared significantly increased during acute infection whereas biologically active tumor necrosis factor was virtually undetectable in serum from infected rats regardless of whether they had been given IFN-γ or not. The prevalence of chronic focal myocarditis in IFN-γ-treated infected rats showed no differences with respect to the one recorded in control-infected counterparts. The inverse CD4/CD8 ratio of spleen and lymph node T cells that usually accompanies chronic infection was reversed by IFN-γ. Mononuclear cells carrying class III-A and I-E molecules, that were found to have increased at both compartments, appeared also modified upon IFN-γ treatment with an overincrease of I-A-positive cells, and a normalization of I-E-bearing cells.     

The Antiarrhythmic Peptide Rotigaptide (ZP123) Increases Connexin 43 Protein Expression in Neonatal Rat Ventricular Cardiomyocytes

Rotigaptide (formerly ZP123) is a novel antiarrhythmic peptide that prevents uncoupling of connexin 43 (Cx43)-mediated, gap junction communication during acute metabolic stress. Since rotigaptide's long-term effects on Cx43 are unknown, we studied its effect on Cx43 protein levels at 24 h in neonatal ventricular myocytes. As determined by Western blot analysis, rotigaptide produced a dose-dependent increase in Cx43 protein expression that reached a maximum level at 100 nM. Furthermore, 100 nM rotigaptide markedly increased Cx43 immunoreactivity and Cx43-positive gap junctions as observed in immunocytochemical studies. Cycloheximide, an inhibitor of protein synthesis, was used to investigate rotigaptide's mechanism of action. Cycloheximide (10 μg/ml) reduced Cx43 protein levels to 39% of vehicle (17 mM ethanol) whereas cotreatment of 10 μg/ml cycloheximide with 100 nM rotigaptide reduced Cx43 protein levels to 56% of vehicle. Our findings suggest that rotigaptide's effect on Cx43 expression is partly due to increased biosynthesis.     

AKIP1 Expression Modulates Mitochondrial Function in Rat Neonatal Cardiomyocytes

A kinase interacting protein 1 (AKIP1) is a molecular regulator of protein kinase A and nuclear factor kappa B signalling. Recent evidence suggests AKIP1 is increased in response to cardiac stress, modulates acute ischemic stress response, and is localized to mitochondria in cardiomyocytes. The mitochondrial function of AKIP1 is, however, still elusive. Here, we investigated the mitochondrial function of AKIP1 in a neonatal cardiomyocyte model of phenylephrine (PE)-induced hypertrophy. Using a seahorse flux analyzer we show that PE stimulated the mitochondrial oxygen consumption rate (OCR) in cardiomyocytes. This was partially dependent on PE mediated AKIP1 induction, since silencing of AKIP1 attenuated the increase in OCR. Interestingly, AKIP1 overexpression alone was sufficient to stimulate mitochondrial OCR and in particular ATP-linked OCR. This was also true when pyruvate was used as a substrate, indicating that it was independent of glycolytic flux. The increase in OCR was independent of mitochondrial biogenesis, changes in ETC density or altered mitochondrial membrane potential. In fact, the respiratory flux was elevated per amount of ETC, possibly through enhanced ETC coupling. Furthermore, overexpression of AKIP1 reduced and silencing of AKIP1 increased mitochondrial superoxide production, suggesting that AKIP1 modulates the efficiency of electron flux through the ETC. Together, this suggests that AKIP1 overexpression improves mitochondrial function to enhance respiration without excess superoxide generation, thereby implicating a role for AKIP1 in mitochondrial stress adaptation. Upregulation of AKIP1 during different forms of cardiac stress may therefore be an adaptive mechanism to protect the heart.     

Exacerbation of Experimental Trypanosoma cruzi Infection in Mice by Concomitant Malaria*

SYNOPSIS. The effect of malaria on the chronic phase of Chagas’disease was investigated in mice. The animals were given Plasmodium berghei-infected red blood cells 2 to 12 months after their initial inoculation with trypomastigotes of 3 different strains of Trypanosoma cruzi (Y, CL and Gilmar). In all the experiments carried out with one of the strains (CL), a somewhat variable but always considerable percentage of mice (average 39%) relapsed in to the acute phase of Chagas’disease. This relapse was characterized by a significant increase in the number of circulating trypomastigotes. Recrudescence was observed also with a 2nd strain of T. cruzi (Gilmar), which is similar in many aspects to the CL strain, e.g. the morphology of blood stages, curve of parasitemia and susceptibility to antibodies in vitro. In mice whose chronic phase was induced by trypomastigotes of the Y strain, malaria infections did not induce a typical acute phase with high parasitemia by T. cruzi. Bloodstream forms of Y parasites differ from those of CL and Gilmar strains morphologically as well as immunologically, i.e. only the Y strain is easily agglutinated and partly inactivated by specific immune serum. In light of this and other known characteristics of the strains used in the present work, the author speculates on mechanisms which allow malaria infections selectively to suppress acquired host resistance to certain strains of T. cruzi.     

Global Metabolomic Profiling of Acute Myocarditis Caused by Trypanosoma cruzi Infection

Chagas disease is caused by Trypanosoma cruzi infection, being cardiomyopathy the more frequent manifestation. New chemotherapeutic drugs are needed but there are no good biomarkers for monitoring treatment efficacy. There is growing evidence linking immune response and metabolism in inflammatory processes and specifically in Chagas disease. Thus, some metabolites are able to enhance and/or inhibit the immune response. Metabolite levels found in the host during an ongoing infection could provide valuable information on the pathogenesis and/or identify deregulated metabolic pathway that can be potential candidates for treatment and being potential specific biomarkers of the disease. To gain more insight into those aspects in Chagas disease, we performed an unprecedented metabolomic analysis in heart and plasma of mice infected with T. cruzi. Many metabolic pathways were profoundly affected by T. cruzi infection, such as glucose uptake, sorbitol pathway, fatty acid and phospholipid synthesis that were increased in heart tissue but decreased in plasma. Tricarboxylic acid cycle was decreased in heart tissue and plasma whereas reactive oxygen species production and uric acid formation were also deeply increased in infected hearts suggesting a stressful condition in the heart. While specific metabolites allantoin, kynurenine and p-cresol sulfate, resulting from nucleotide, tryptophan and phenylalanine/tyrosine metabolism, respectively, were increased in heart tissue and also in plasma. These results provide new valuable information on the pathogenesis of acute Chagas disease, unravel several new metabolic pathways susceptible of clinical management and identify metabolites useful as potential specific biomarkers for monitoring treatment and clinical severity in patients.     

Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes

The ability of azobenzene trimethylammonium bromide (azoTAB) to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav), calcium (ICav), and potassium (IKv) currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na⁺) and calcium (Ca²⁺) currents and potentiation of net potassium (K⁺) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential.     

Bile Acid-Induced Arrhythmia Is Mediated by Muscarinic M2 Receptors in Neonatal Rat Cardiomyocytes

Background

Intrahepatic cholestasis of pregnancy (ICP) is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that bile acid taurocholate (TC), which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM). Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart.

Methods and Results

Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M₂ receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M₂ receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters.

Conclusion

We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M₂ receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.     

A Recombinant Protein Based on Trypanosoma cruzi P21 Enhances Phagocytosis

Background

P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His₆) on inflammatory macrophages during phagocytosis.

Findings

Our results showed that P21-His₆ acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent onthe PI3-kinase signaling pathway.

Conclusions

Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His₆ represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.     

High Fat Diet Modulates Trypanosoma cruzi Infection Associated Myocarditis

Background

Trypanosoma cruzi, the causative agent of Chagas disease, has high affinity for lipoproteins and adipose tissue. Infection results in myocarditis, fat loss and alterations in lipid homeostasis. This study was aimed at analyzing the effect of high fat diet (HFD) on regulating acute T. cruzi infection-induced myocarditis and to evaluate the effect of HFD on lipid metabolism in adipose tissue and heart during acute T. cruzi infection.

Methodology/Principal Findings

CD1 mice were infected with T. cruzi (Brazil strain) and fed either a regular control diet (RD) or HFD for 35 days following infection. Serum lipid profile, tissue cholesterol levels, blood parasitemia, and tissue parasite load were analyzed to evaluate the effect of diet on infection. MicroPET and MRI analysis were performed to examine the morphological and functional status of the heart during acute infection. qPCR and immunoblot analysis were carried out to analyze the effect of diet on the genes involved in the host lipid metabolism during infection. Oil red O staining of the adipose tissue demonstrated reduced lipolysis in HFD compared to RD fed mice. HFD reduced mortality, parasitemia and cardiac parasite load, but increased parasite load in adipocytes. HFD decreased lipolysis during acute infection. Both qPCR and protein analysis demonstrated alterations in lipid metabolic pathways in adipose tissue and heart in RD fed mice, which were further modulated by HFD. Both microPET and MRI analyses demonstrated changes in infected RD murine hearts which were ameliorated by HFD.

Conclusion/Significance

These studies indicate that Chagasic cardiomyopathy is associated with a cardiac lipidpathy and that both cardiac lipotoxicity and adipose tissue play a role in the pathogenesis of Chagas disease. HFD protected mice from T. cruzi infection-induced myocardial damage most likely due to the effects of HFD on both adipogenesis and T. cruzi infection-induced cardiac lipidopathy.     

赤芍总苷对培养乳鼠心肌细胞损伤的保护作用

为了探讨赤芍总苷对心肌细胞损伤的保护作用,以异丙基肾上腺素加入培养的乳鼠心肌细胞造成缺血缺氧损伤模型,对比分析正常对照组、药物损伤组、辅酶Q10阳性对照组、以及不同剂量赤芍总苷保护组的细胞形态学、心肌酶谱等指标、结果显示：损伤组细胞搏动加速,存活率下降,GOT、LDH、CK等3种心肌酶活力均显著升高;而辅酶Q10组和赤芍总苷组上述指标都有不同程度的改善,其中高剂量赤芍总苷组的保护作用优于阳性对照组．证明赤芍总苷对异丙基肾上腺素造成的培养乳鼠心肌细胞损伤具有保护作用,并且呈现剂量依赖关系．     

Galectin-1 Prevents Infection and Damage Induced by Trypanosoma cruzi on Cardiac Cells

Background

Chronic Chagas cardiomyopathy caused by Trypanosoma cruzi is the result of a pathologic process starting during the acute phase of parasite infection. Among different factors, the specific recognition of glycan structures by glycan-binding proteins from the parasite or from the mammalian host cells may play a critical role in the evolution of the infection.

Methodology and Principal Findings

Here we investigated the contribution of galectin–1 (Gal–1), an endogenous glycan-binding protein abundantly expressed in human and mouse heart, to the pathophysiology of T. cruzi infection, particularly in the context of cardiac pathology. We found that exposure of HL–1 cardiac cells to Gal–1 reduced the percentage of infection by two different T. cruzi strains, Tulahuén (TcVI) and Brazil (TcI). In addition, Gal–1 prevented exposure of phosphatidylserine and early events in the apoptotic program by parasite infection on HL–1 cells. These effects were not mediated by direct interaction with the parasite surface, suggesting that Gal–1 may act through binding to host cells. Moreover, we also observed that T. cruzi infection altered the glycophenotype of cardiac cells, reducing binding of exogenous Gal–1 to the cell surface. Consistent with these data, Gal–1 deficient (Lgals1 ^-/-) mice showed increased parasitemia, reduced signs of inflammation in heart and skeletal muscle tissues, and lower survival rates as compared to wild-type (WT) mice in response to intraperitoneal infection with T. cruzi Tulahuén strain.

Conclusion/Significance

Our results indicate that Gal–1 modulates T. cruzi infection of cardiac cells, highlighting the relevance of galectins and their ligands as regulators of host-parasite interactions.     

Trypanosoma cruzi Infection of Cultured Adipocytes Results in an Inflammatory Phenotype

Infection with Trypanosoma cruzi, the etiologic agent of Chagas disease is accompanied by an intense inflammatory reaction. Our laboratory group has identified adipose tissue as one of the major sites of inflammation during disease progression. Because adipose tissue is composed of many cell types, we were interested in investigating whether the adipocyte per se was a source of inflammatory mediators in this infection. Cultured adipocytes were infected with the Tulahuen strain of T. cruzi for 48–96 h. Immunoblot and quantitative PCR (qPCR) analyses demonstrated an increase in the expression of proinflammatory cytokines and chemokines, including interleukin (IL)‐1β, interferon‐γ, tumor necrosis factor‐α, CCL2, CCL5, and CXCL10 as well as an increase in the expression of Toll‐like receptors‐2 and 9 and activation of the notch pathway. Interestingly, caveolin‐1 expression was reduced while cyclin D1 and extracellular signal‐regulated kinase (ERK) expression was increased. The expression of PI3kinase and the activation of AKT (phosphorylated AKT) were increased suggesting that infection may induce components of the insulin/IGF‐1 receptor cascade. There was an infection‐associated decrease in adiponectin and peroxisome proliferator‐activated receptor‐γ (PPAR‐γ). These data provide a mechanism for the increase in the inflammatory phenotype that occurs in T. cruzi‐infected adipocytes. Overall, these data implicate the adipocyte as an important target of T. cruzi, and one which contributes significantly to the inflammatory response observed in Chagas disease.     

Prostanoid Receptors Involved in Regulation of the Beating Rate of Neonatal Rat Cardiomyocytes

Although prostanoids are known to be involved in regulation of the spontaneous beating rate of cultured neonatal rat cardiomyocytes, the various subtypes of prostanoid receptors have not been investigated in detail. In our experiments, prostaglandin (PG)F_2α and prostanoid FP receptor agonists (fluprostenol, latanoprost and cloprostenol) produced a decrease in the beating rate. Two prostanoid IP receptor agonists (iloprost and beraprost) induced first a marked drop in the beating rate and then definitive abrogation of beating. In contrast, the prostanoid DP receptor agonists (PGD₂ and BW245C) and TP receptor agonists (U-46619) produced increases in the beating rate. Sulprostone (a prostanoid EP₁ and EP₃ receptor agonist) induced marked increases in the beating rate, which were suppressed by SC-19220 (a selective prostanoid EP₁ antagonist). Butaprost (a selective prostanoid EP₂ receptor agonist), misoprostol (a prostanoid EP₂ and EP₃ receptor agonist), 11-deoxy-PGE₁ (a prostanoid EP₂, EP₃ and EP₄ receptor agonist) did not alter the beating rate. Our results strongly suggest that prostanoid EP₁ receptors are involved in positive regulation of the beating rate. Prostanoid EP₁ receptor expression was confirmed by western blotting with a selective antibody. Hence, neonatal rat cardiomyocytes express both prostanoid IP and FP receptors (which negatively regulate the spontaneous beating rate) and prostanoid TP, DP₁ and EP₁ receptors (which positively regulate the spontaneous beating rate).