ß-Adrenergic signaling drives structural and functional maturation of mouse cardiomyocytes.

Cardiac maturation represents the last phase of heart development and is characterized by morphofunctional alterations that optimize the heart for efficient pumping. Its understanding provides important insights into cardiac regeneration therapies. Recent evidence implies that adrenergic signals are involved in the regulation of cardiac maturation, but the mechanistic underpinnings involved in this process are poorly understood. Herein, we explored the role of ß-adrenergic receptor (ß-AR) activation in determining structural and functional components of cardiomyocyte maturation. Temporal characterization of tyrosine hydroxylase and norepinephrine levels in the mouse heart revealed that sympathetic innervation develops during the first 3 wk of life, concurrent with the rise in ß-AR expression. To assess the impact of adrenergic inhibition on maturation, we treated mice with propranolol, isolated cardiomyocytes, and evaluated morphofunctional parameters. Propranolol treatment reduced heart weight, cardiomyocyte size, and cellular shortening, while it increased the pool of mononucleated myocytes, resulting in impaired maturation. No changes in t-tubules were observed in cells from propranolol mice. To establish a causal link between ß-AR signaling and cardiomyocyte maturation, mice were subjected to sympathectomy, followed or not by restoration with isoproterenol treatment. Cardiomyocytes from sympathectomyzed mice recapitulated the salient immaturity features of propranolol-treated mice, with the additional loss of t-tubules. Isoproterenol rescued the maturation deficits induced by sympathectomy, except for the t-tubule alterations. Our study identifies the ß-AR stimuli as a maturation promoting signal and implies that this pathway can be modulated to improve cardiac regeneration therapies.NEW & NOTEWORTHY Maturation involves a series of morphofunctional alterations vital to heart development. Its regulatory mechanisms are only now being unveiled. Evidence implies that adrenergic signaling regulates cardiac maturation, but the mechanisms are poorly understood. To address this point, we blocked ß-ARs or performed sympathectomy followed by rescue experiments with isoproterenol in neonatal mice. Our study identifies the ß-AR stimuli as a maturation signal for cardiomyocytes and highlights the importance of this pathway in cardiac regeneration therapies.

The Mas agonist CGEN-856S prevents Ang II induced cardiomyocyte hypertrophy via nitric oxide production.

With the previous knowledge of the cardioprotective effects of the Angiotensin-(1-7) axis, a agonist of Mas receptor has been described, the CGEN-856S. This peptide is more stable than Ang-(1-7), and has a low binding affinity to Angiotensin II receptors. Although the cardioprotective effects of CGEN-856S were previously shown in vivo, the mechanisms behind its effects are still unknown. Here, we employed a combination of molecular biology, confocal microscopy, and genetically modified mouse with Mas deletion to investigate the CGEN-856S protective signaling in cardiomyocytes. In isolated adult ventricular myocytes, CGEN-856S induced an increase in nitric oxide (NO) production which was absent in cells from Mas knockout mice. Using western blot, we observed a significant increase in phosphorylation of AKT after treatment with CGEN-856S. In addition, CGEN-856S prevented the Ang II induced hypertrophy and the nuclear translocation of GRK5 in a culture model of rat neonatal cardiomyocytes. Blockage of Mas receptor and inhibition of the NO synthase abolished the effects of CGEN-856S on Ang II treated cardiomyocytes. In conclusion, we show that CGEN-856S acting via receptor Mas induces NO raise to block Ang II induced cardiomyocyte hypertrophy. These results indicate that CGEN-856S acts very similarly to Ang-(1-7) in cardiac myocytes, highlighting its therapeutic potential for treating cardiovascular diseases.

Necrotic cardiac myocytes skew macrophage polarization towards a classically activated phenotype.

Necrotic and dying cells release damage-associated molecular patterns (DAMPs) that can initiate sterile inflammatory responses in the heart. Although macrophages are essential for myocardial repair and regeneration, the effect of DAMPs on macrophage activation remains unclear. To address this gap in knowledge we studied the effect of necrotic cardiac myocyte extracts on primary peritoneal macrophage (PPM) cultures in vitro. We first performed unbiased transcriptomic profiling with RNA-sequencing of PPMs cultured for up to 72 hours in the presence and absence of: 1) necrotic cell extracts (NCEs) from necrotic cardiac myocytes in order to mimic the release of DAMPs; 2) lipopolysaccharide (LPS), which is known to polarize macrophages towards a classically activated phenotype and 3) Interleukin-4 (IL-4), which is known to promote polarization of macrophages towards an alternatively activated phenotype. NCEs provoke changes in differential gene expression (DEGs) that had considerable overlap with LPS-induced changes, suggesting that NCEs promote macrophage polarization towards a classically activated phenotype. Treating NCEs with proteinase-K abolished the effects of NCEs on macrophage activation, whereas NCE treatment with DNase and RNase did not affect macrophage activation. Stimulation of macrophage cultures with NCEs and LPS resulted in a significant increase in macrophage phagocytosis and interleukin-1ß secretion, whereas treatment with IL-4 had no significant effect on phagocytosis and interleukin-1ß. Taken together, our findings suggest that proteins released from necrotic cardiac myocytes are sufficient to skew the polarization of macrophages towards a classically activated phenotype.

Refining the reproducibility of a murine model of stress-induced reversible cardiomyopathy.

Despite the many advantages of isoproterenol (Iso)-induced models of cardiomyopathy, the extant literature suggests that the reproducibility of the Iso-induced stress cardiomyopathy phenotype varies considerably depending on the dose of Iso used, the mode of administration of Iso (subcutaneous vs. intraperitoneal), and the species of the animal that is being studied. Recently, we have shown that a single injection of Iso into female C57BL/6J mice provokes transient myocardial injury that is characterized by a brisk release of troponin I within 1 h, as well as a self-limited myocardial inflammatory response that is associated with increased myocardial tissue edema, inferoapical regional left ventricular (LV) wall motion abnormalities, and a transient decrease in global LV function, which were completely recovered by day 7 after the Iso injection (i.e., stress-induced reversible cardiomyopathy). Here we expand upon this initial report in this model by demonstrating important sexually dimorphic differences in the response to Iso-induced tissue injury, the ensuing myocardial inflammatory response, and changes in LV structure and function. We also provide information with respect to enhancing the reproducibility in this model by optimizing animal welfare during the procedure. The acute Iso-induced myocardial injury model provides a low-cost, relatively high-throughput small-animal model that mimics human disease (e.g., Takotsubo cardiomyopathy). Given that the model can be performed in different genetic backgrounds, as well as different experimental conditions, the acute Iso injury model should provide the cardiovascular community with a valuable nonsurgical animal model for understanding the myocardial response to tissue injury.NEW & NOTEWORTHY The present study highlights the importance of sexual dimorphism with respect to isoproterenol injury, as well as the importance of animal handling and welfare to obtain reproducible results from investigator to investigator. Based on serial observations of animal recovery (locomotor activity and grooming behavior), troponin I release, and inflammation, we identified that the method used to restrain the mice for the intraperitoneal injection was the single greatest source of variability in this model.

Neuronal cholinergic signaling constrains norepinephrine activity in the heart.

It is well known that cholinergic hypofunction contributes to cardiac pathology, yet, the mechanisms involved remain unclear. Our previous study has shown that genetically engineered model of cholinergic deficit, the vesicular acetylcholine transporter knockdown homozygous (VAChT KDHOM) mice, exhibit pathological cardiac remodeling and a gradual increase in cardiac mass with aging. Given that an increase in cardiac mass is often caused by adrenergic hyperactivity, we hypothesized that VAChT KDHOM mice might have an increase in cardiac norepinephrine (NE) levels. We thus investigated the temporal changes in NE content in the heart from 3-, 6-, and 12-mo-old VAChT mutants. Interestingly, mice with cholinergic hypofunction showed a gradual elevation in cardiac NE content, which was already increased at 6 mo of age. Consistent with this finding, 6-mo-old VAChT KDHOM mice showed enhanced sympathetic activity and a greater abundance of tyrosine hydroxylase positive sympathetic nerves in the heart. VAChT mutants exhibited an increase in peak calcium transient, and mitochondrial oxidative stress in cardiomyocytes along with enhanced G protein-coupled receptor kinase 5 (GRK5) and nuclear factor of activated T-cells (NFAT) staining in the heart. These are known targets of adrenergic signaling in the cell. Moreover, vagotomized-mice displayed an increase in cardiac NE content confirming the data obtained in VAChT KDHOM mice. Establishing a causal relationship between acetylcholine and NE, VAChT KDHOM mice treated with pyridostigmine, a cholinesterase inhibitor, showed reduced cardiac NE content, rescuing the phenotype. Our findings unveil a yet unrecognized role of cholinergic signaling as a modulator of cardiac NE, providing novel insights into the mechanisms that drive autonomic imbalance.

B cells modulate the expression of MHC-II on cardiac CCR2- macrophages.

The uninjured murine heart contains a heterogeneous population of macrophages with disparate ontogenies and functions. These macrophages are often associated with blood vessels and can be subclassified based on the expression of CC chemokine receptor 2 (CCR2) and major histocompatibility complex class II (MHC-II). The biological cues that modulate these macrophage pool subpopulations have not been completely identified. It has been recently shown that a sub-population of circulating naïve B cells adheres to the myocardial microvasculature. We hypothesized that B cells might modulate the phenotype of myocardial macrophages. To test this hypothesis, we analyzed both the relative location of B cells and macrophages in myocardial histological section and the prevalence of myocardial macrophage subsets in hearts from B cell-deficient mice (µMT) and mice depleted of B cells through administration of an anti-CD20 antibody. We found that B cells pause in the microvasculature in proximity of macrophages and modulate the number of myocardial CCR2-MHC-IIhigh cells. Through in vitro studies we found that this is likely the result of a paracrine effect of B cells on the expression of MHC-II in CCR2- cells. These results reveal an unexpected relationship between B cells and resident macrophages and, highlighting a direct intramyocardial effect of circulating B cells, challenge the currently held belief that naïve recirculating B lymphocytes merely shuttle between lymphoid stations.

Protective and anti-inflammatory effects of acetylcholine in the heart.

The innate and adaptive immune systems play an important role in the development of cardiac diseases. Therefore, it has become critical to identify molecules that can modulate inflammation in the injured heart. In this regard, activation of the cholinergic system in animal models of heart disease has been shown to exert protective actions that include immunomodulation of cardiac inflammation. In this mini-review, we briefly present our current understanding on the cardiac cellular sources of acetylcholine (ACh) (neuronal vs. nonneuronal), followed by a discussion on its contribution to the regulation of inflammatory cells. Although the mechanism behind ACh-mediated protection still remains to be fully elucidated, the beneficial immunomodulatory role of the cholinergic signaling emerges as a potential key regulator of cardiac inflammation.

Increased cholinergic activity under conditions of low estrogen leads to adverse cardiac remodeling.

Cholinesterase inhibitors are used in postmenopausal women for the treatment of neurodegenerative diseases. Despite their widespread use in the clinical practice, little is known about the impact of augmented cholinergic signaling on cardiac function under reduced estrogen conditions. To address this gap, we subjected a genetically engineered murine model of systemic vesicular acetylcholine transporter overexpression (Chat-ChR2) to ovariectomy and evaluated cardiac parameters. Left-ventricular function was similar between Chat-ChR2 and wild-type (WT) mice. Following ovariectomy, WT mice showed signs of cardiac hypertrophy. Conversely, ovariectomized (OVX) Chat-ChR2 mice evolved to cardiac dilation and failure. Transcript levels for cardiac stress markers atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) were similarly upregulated in WT/OVX and Chat-ChR2/OVX mice. 17ß-Estradiol (E2) treatment normalized cardiac parameters in Chat-ChR2/OVX to the Chat-ChR2/SHAM levels, providing a link between E2 status and the aggravated cardiac response in this model. To investigate the cellular basis underlying the cardiac alterations, ventricular myocytes were isolated and their cellular area and contractility were assessed. Myocytes from WT/OVX mice were wider than WT/SHAM, an indicative of concentric hypertrophy, but their fractional shortening was similar. Conversely, Chat-ChR2/OVX myocytes were elongated and presented contractile dysfunction. E2 treatment again prevented the structural and functional changes in Chat-ChR2/OVX myocytes. We conclude that hypercholinergic mice under reduced estrogen conditions do not develop concentric hypertrophy, a critical compensatory adaptation, evolving toward cardiac dilation and failure. This study emphasizes the importance of understanding the consequences of cholinesterase inhibition, used clinically to treat dementia, for cardiac function in postmenopausal women.

Proteomic Signatures of Heart Failure in Relation to Left Ventricular Ejection Fraction.

BACKGROUND: There is a growing recognition of the inherent limitations of the use of the left ventricular ejection fraction (LVEF) to accurately phenotype patients with heart failure (HF). OBJECTIVES: The authors sought to identify unique proteomic signatures for patients with HF with reduced ejection fraction (HFrEF), HF with a midrange LVEF (HFmrEF), and HF with preserved ejection fraction (HFpEF), as well as to identify molecular differences between patients with ischemic and nonischemic HF. METHODS: We used high-content aptamer-based proteomics technology (SOMAscan) to interrogate the blood proteome of age- and sex-matched patients with HF within different LVEF groups. RESULTS: Within the Washington University Heart Failure Registry, we identified age/sex-matched patients within 3 LVEF categories: HFrEF (LVEF <40%), HFmrEF (LVEF 40% to 50%), and HFpEF (LVEF >50%). We found that patients with HFrEF, HFmrEF, and HFpEF had unique variations in circulating proteins that reflected distinct biological pathophysiologies. Bioinformatics analysis revealed that there were biological themes that were unique to patients with HFrEF, HFpEF, or HFmrEF. Comparative analyses of patients with HFmrEF with improved LVEF and patients with HFmrEF with unchanged LVEF revealed marked differences between these 2 patient populations and indicated that patients with recovered LVEF are more similar to patients with HFpEF than to patients with HFrEF. Moreover, there were marked differences in the proteomic signatures of patients with ischemic and nonischemic HF. CONCLUSIONS: Viewed together, these findings suggest that it may be possible to use high-content multiplexed proteomics assays in combination with the clinical assessment of LVEF to more accurately identify clinical phenotypes of patients with HF.

Developmental changes in myocardial B cells mirror changes in B cells associated with different organs.

The naive heart harbors a population of intravascular B cells that make close contact with the cardiac microvasculature. However, the timing of their appearance and their organ specificity remain unknown. To address this knowledge gap, we performed a systematic analysis of B cells isolated from the myocardium and other organs, from embryonic life to adulthood. We found that the phenotype of myocardial B cells changed dynamically during development. While neonatal heart B cells were mostly CD11b+ and CD11b- CD21-CD23-, adult B cells were predominantly CD11b-CD21+CD23+. Histological analysis and intravital microscopy of lung and liver showed that organ-associated B cells in contact with the microvascular endothelium were not specific to the heart. Flow cytometric analysis of perfused hearts, livers, lungs, and spleen showed that the dynamic changes in B cell subpopulations observed in the heart during development mirrored changes observed in the other organs. Single cell RNA sequencing (scRNAseq) analysis of B cells showed that myocardial B cells were part of a larger population of organ-associated B cells that had a distinct transcriptional profile. These findings broaden our understanding of the biology of myocardial-associated B cells and suggest that current models of the dynamics of naive B cells during development are incomplete.

The Emerging Role of B Lymphocytes in Cardiovascular Disease.

B cells are traditionally known for their ability to produce antibodies in the context of adaptive immune responses. However, over the last decade B cells have been increasingly recognized as modulators of both adaptive and innate immune responses, as well as players in an important role in the pathogenesis of a variety of human diseases. Here, after briefly summarizing our current understanding of B cell biology, we present a systematic review of the literature from both animal models and human studies that highlight the important role that B lymphocytes play in cardiac and vascular disease. While many aspects of B cell biology in the vasculature and, to an even greater extent, in the heart remain unclear, B cells are emerging as key regulators of cardiovascular adaptation to injury.

Reappraising the role of inflammation in heart failure.

The observation that heart failure with reduced ejection fraction is associated with elevated circulating levels of pro-inflammatory cytokines opened a new area of research that has revealed a potentially important role for the immune system in the pathogenesis of heart failure. However, until the publication in 2019 of the CANTOS trial findings on heart failure outcomes, all attempts to target inflammation in the heart failure setting in phase III clinical trials resulted in neutral effects or worsening of clinical outcomes. This lack of positive results in turn prompted questions on whether inflammation is a cause or consequence of heart failure. This Review summarizes the latest developments in our understanding of the role of the innate and adaptive immune systems in the pathogenesis of heart failure, and highlights the results of phase III clinical trials of therapies targeting inflammatory processes in the heart failure setting, such as anti-inflammatory and immunomodulatory strategies. The most recent of these studies, the CANTOS trial, raises the exciting possibility that, in the foreseeable future, we might be able to identify those patients with heart failure who have a cardio-inflammatory phenotype and will thus benefit from therapies targeting inflammation.

Myocardial B cells are a subset of circulating lymphocytes with delayed transit through the heart.

Current models of B lymphocyte biology posit that B cells continuously recirculate between lymphoid organs, without accumulating in peripheral healthy tissues. Nevertheless, B lymphocytes are one of the most prevalent leukocyte populations in the naive murine heart. To investigate this apparent inconsistency in the literature, we conducted a systematic analysis of myocardial B cell ontogeny, trafficking dynamics, histology, and gene expression patterns. We found that myocardial B cells represent a subpopulation of circulating B cells that make close contact with the microvascular endothelium of the heart and arrest their transit as they pass through the heart. The vast majority (>95%) of myocardial B cells remain intravascular, whereas few (<5%) myocardial B cells cross the endothelium into myocardial tissue. Analyses of mice with B cell deficiency or depletion indicated that B cells modulate the myocardial leukocyte pool composition. Analysis of B cell-deficient animals suggested that B cells modulate myocardial growth and contractility. These results transform our current understanding of B cell recirculation in the naive state and reveal a previously unknown relationship between B cells and myocardial physiology. Further work will be needed to assess the relevance of these findings to other organs.

Immunomodulatory role of non-neuronal cholinergic signaling in myocardial injury.

Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the non-neuronal cholinergic system is not well understood. To address the immunomodulatory role of the non-neuronal cholinergic system in the heart we used a previously validated diphtheria toxin (DT)-induced cardiomyocyte ablation model (Rosa26-DTMlc2v-Cre mice). DT-injected Rosa26-DTMlc2v-Cre mice were treated with diluent or Pyridostigmine Bromide (PYR), a reversible cholinesterase inhibitor. PYR treatment resulted in increased survival and decreased numbers of MHC-IIlowCCR2+ macrophages in DT-injected Rosa26-DTMlc2v-Cre mice compared to diluent treated Rosa26-DTMlc2v-Cre mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DTMlc2v-Cre mice with a transgenic mouse line (Chat-ChR2) that constitutively overexpresses the vesicular acetylcholine transporter (VAChT) resulted in decreased expression of Ccl2/7 mRNA and decreased numbers of CD68+ cells in DT-injured Rosa26-DTMlc2v-Cre/Chat-ChR2 mouse hearts, consistent with the pharmacologic studies with PYR. In vitro studies with cultures of LPS-stimulated peritoneal macrophages revealed a concentration-dependent reduction in CCL2 secretion following stimulation with ACh, nicotine and muscarine. Viewed together, these findings reveal a previously unappreciated immunomodulatory role for the non-neuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury.

Modulation of subsets of cardiac B lymphocytes improves cardiac function after acute injury.

Despite the long-standing recognition that the immune response to acute myocardial injury contributes to adverse left ventricular (LV) remodeling, it has not been possible to effectively target this clinically. Using 2 different in vivo models of acute myocardial injury, we show that pirfenidone confers beneficial effects in the murine heart through an unexpected mechanism that depends on cardiac B lymphocytes. Naive hearts contained a large population of CD19+CD11b-CD23-CD21-IgD+IgMlo lymphocytes, and 2 smaller populations of CD19+CD11b+ B1a and B1b cells. In response to tissue injury, there was an increase in neutrophils, monocytes, macrophages, as well as an increase in CD19+ CD11b- B lymphocytes. Treatment with pirfenidone had no effect on the number of neutrophils, monocytes, or macrophages, but decreased CD19+CD11b- lymphocytes. B cell depletion abrogated the beneficial effects of pirfenidone. In vitro studies demonstrated that stimulation with lipopolysaccharide and extracts from necrotic cells activated CD19+ lymphocytes through a TIRAP-dependent pathway. Treatment with pirfenidone attenuated this activation of B cells. These findings reveal a previously unappreciated complexity of myocardial B lymphocytes within the inflammatory infiltrate triggered by cardiac injury and suggest that pirfenidone exerts beneficial effects in the heart through a unique mechanism that involves modulation of cardiac B lymphocytes.

Alamandine acts via MrgD to induce AMPK/NO activation against ANG II hypertrophy in cardiomyocytes.

The renin-angiotensin system (RAS) plays a pivotal role in the pathogenesis of cardiovascular diseases. New members of this system have been characterized and shown to have biologically relevant actions. Alamandine and its receptor MrgD are recently identified components of RAS. In the cardiovascular system, alamandine actions included vasodilation, antihypertensive, and antifibrosis effects. Currently, the actions of alamandine on cardiomyocytes are unknown. Here our goal was twofold: 1) to unravel the signaling molecules activated by the alamandine/MrgD axis in cardiomyocytes; and 2) to evaluate the ability of this axis to prevent angiotensin II (ANG II)-induced hypertrophy. In cardiomyocytes from C57BL/6 mice, alamandine treatment induced an increase in nitric oxide (NO) production, which was blocked by d-Pro7-ANG-(1-7), a MrgD antagonist. This NO rise correlated with increased phosphorylation of AMPK. Alamandine-induced NO production was preserved in Mas-/- myocytes and lost in MrgD-/- cells. Binding of fluorescent-labeled alamandine was observed in wild-type cells, but it was dramatically reduced in MrgD-/- myocytes. We also assessed the consequences of prolonged alamandine exposure to cultured neonatal rat cardiomyocytes (NRCMs) treated with ANG II. Treatment of NRCMs with alamandine prevented ANG II-induced hypertrophy. Moreover, the antihypertrophic actions of alamandine were mediated via MrgD and NO, since they could be prevented by d-Pro7-ANG-(1-7) or inhibitors of NO synthase or AMPK. ß-Alanine, a MrgD agonist, recapitulated alamandine's cardioprotective effects in cardiomyocytes. Our data show that alamandine via MrgD induces AMPK/NO signaling to counterregulate ANG II-induced hypertrophy. These findings highlight the therapeutic potential of the alamandine/MrgD axis in the heart.

Exercise reestablishes autophagic flux and mitochondrial quality control in heart failure.

We previously reported that facilitating the clearance of damaged mitochondria through macroautophagy/autophagy protects against acute myocardial infarction. Here we characterize the impact of exercise, a safe strategy against cardiovascular disease, on cardiac autophagy and its contribution to mitochondrial quality control, bioenergetics and oxidative damage in a post-myocardial infarction-induced heart failure animal model. We found that failing hearts displayed reduced autophagic flux depicted by accumulation of autophagy-related markers and loss of responsiveness to chloroquine treatment at 4 and 12 wk after myocardial infarction. These changes were accompanied by accumulation of fragmented mitochondria with reduced O2 consumption, elevated H2O2 release and increased Ca2+-induced mitochondrial permeability transition pore opening. Of interest, disruption of autophagic flux was sufficient to decrease cardiac mitochondrial function in sham-treated animals and increase cardiomyocyte toxicity upon mitochondrial stress. Importantly, 8 wk of exercise training, starting 4 wk after myocardial infarction at a time when autophagy and mitochondrial oxidative capacity were already impaired, improved cardiac autophagic flux. These changes were followed by reduced mitochondrial number:size ratio, increased mitochondrial bioenergetics and better cardiac function. Moreover, exercise training increased cardiac mitochondrial number, size and oxidative capacity without affecting autophagic flux in sham-treated animals. Further supporting an autophagy mechanism for exercise-induced improvements of mitochondrial bioenergetics in heart failure, acute in vivo inhibition of autophagic flux was sufficient to mitigate the increased mitochondrial oxidative capacity triggered by exercise in failing hearts. Collectively, our findings uncover the potential contribution of exercise in restoring cardiac autophagy flux in heart failure, which is associated with better mitochondrial quality control, bioenergetics and cardiac function.

Absence of suppressor of cytokine signaling 2 turns cardiomyocytes unresponsive to LIF-dependent increases in Ca2+ levels.

Little is known regarding the role of suppressor of cytokine signaling (SOCS) in the control of cytokine signaling in cardiomyocytes. We investigated the consequences of SOCS2 ablation for leukemia inhibitory factor (LIF)-induced enhancement of intracellular Ca2+ ([Ca2+]i) transient by performing experiments with cardiomyocytes from SOCS2-knockout (ko) mice. Similar levels of SOCS3 transcripts were seen in cardiomyocytes from wild-type and SOCS2-ko mice, while SOCS1 mRNA was reduced in SOCS2-ko. Immunoprecipitation experiments showed increased SOCS3 association with gp130 receptor in SOCS2-ko myocytes. Measurements of Ca2+ in wild-type myocytes exposed to LIF showed a significant increase in the magnitude of the Ca2+ transient. This change was absent in LIF-treated SOCS2-ko cells. LIF activation of ERK and STAT3 was observed in both wild-type and SOCS2-ko cells, indicating that in SOCS2-ko, LIF receptors were functional, despite the lack of effect in the Ca2+ transient. In wild-type cells, LIF-induced increase in [Ca2+]i and phospholamban Thr17 [PLN(Thr17)] phosphorylation was inhibited by KN-93, indicating a role for CaMKII in LIF-induced Ca2+ raise. LIF-induced phosphorylation of PLN(Thr17) was abrogated in SOCS2-ko myocytes. In wild-type cardiomyocytes, LIF treatment increased L-type Ca2+ current (ICa,L), a key activator of CaMKII in response to LIF. Conversely, SOCS2-ko myocytes failed to activate ICa,L in response to LIF, providing a rationale for the lack of LIF effect on Ca2+ transient. Our data show that absence of SOCS2 turns cardiomyocytes unresponsive to LIF-induced [Ca2+] raise, indicating that endogenous levels of SOCS2 are crucial for full activation of LIF signaling in the heart.