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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), as well as 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 hour, and 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 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 and neurogenic stunned myocardium). 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 non-surgical animal model for understanding the myocardial response to tissue injury.

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 naive 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 naive recirculating B lymphocytes merely shuttle between lymphoid stations.

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.

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 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.

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. Inflammation has an important role in the pathogenesis of acute and chronic heart failure. This Review summarizes the latest findings on 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 this condition, such as anti-inflammatory and immunomodulatory strategies.

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. 17β-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.

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-II(high) 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.

Background: The murine myocardium contains distinct subsets of macrophages with disparate roles that can be defined by expression levels of CCR2 and MHC-II. The factors that modulate the composition of the myocardial macrophage pool in the naïve heart are not completely known. B cells have been recently shown to modulate the prevalence of T cells in the heart but the relationship between B cells and myocardial macrophage subsets remains unknown. Objective: To test the hypothesis that myocardial-associated B cells modulate the composition of the myocardial macrophage pool. Methods and Results: Hearts from B cell deficient (μMT) or B cell depleted (treated with Anti-CD20 antibody) mice and age/sex matched controls were analyzed via flow cytometry at post-natal day 14 and soon after weaning. Absence of B cells was consistently associated with a decrease in the prevalence of CCR2 - MHC-II high resident macrophages. Immunofluorescence analysis of the heart showed that the myocardial-associated B cells were frequently in close proximity of CD68+ myocardial macrophages. In vitro co-culture of myocardial-derived CD64+ macrophages with B cells or with B cell-conditioned media showed that B cells fostered an increase in the prevalence of CCR2 - MHC-II high macrophages through a direct paracrine effect. In vivo 10X single cells sequencing and flow cytometric analysis together with in vitro ELISA and antibody mediate blocking suggested that the paracrine effect of B cells on myocardial macrophages is mediated, at least in part, by IL-16 secretion. Conclusion: Myocardial-associated B cells modulate the phenotype of myocardial resident macrophages. This is likely the result of a direct paracrine effect mediated, at least in part, by IL-16. These findings describe for the first time a relationship between circulating naïve B cells and tissue resident macrophages and unveil a previously unappreciated relationship between lymphoid and myeloid myocardial cells.

Cryptome is as a subset of a given proteome containing bioactive cryptides embedded in larger peptides or proteins. We pinpointed a striking sequence similarity between two peptides from the Tityus serrulatus venom: Ts10 (KKDGYPVEYDRAY) and the N-terminal of Ts3 (KKDGYPVEYDNCAY). Ts3 (former Tityustoxin or TsIV) is an α-neurotoxin acting on voltage-gated sodium channels while Ts10 (former Peptide T) is a bradykinin-potentiating peptide and was originally reported as inhibitor of the angiotensin-converting enzyme (ACEi). Thus, the goal of this study was to evaluate whether such peptide hidden in the N-terminal of Ts3 (Ts31-14[C12S]) was able to mimic known effects of Ts10 as well as to expand the current knowledge of the vascular effects and molecular targets of these peptides. Similar to Ts10, Ts31-14[C12S] was able to potentiate the hypotensive effect of bradykinin (BK). However, none of these peptides was able to induce a long-lasting BK-potentiating effect, suggesting that this effect may not be their main biological outcome. On the other hand, we report that Ts10 and mainly Ts31-14[C12S] induced a strong vasodilation effect depending on the presence of functional endothelium and nitric oxide (NO) production. Unlike previously reported, Ts10 was not able to inhibit ACE activity (similar result was observed for Ts31-14[C12S]). On the other hand, we report that Ts31-14[C12S] induces vasodilation via the activation of muscarinic acetylcholine receptors (mAChRs) M2 and M3 while only the activation of mAChR M2 seems to be required for Ts10-induced vasodilation.

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.

Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the nonneuronal cholinergic system is not well understood. To address the immunomodulatory role of the nonneuronal cholinergic system in the heart, we used a previously validated diphtheria toxin–induced (DT-induced) cardiomyocyte ablation model (Rosa26-DT(Mlc2v-Cre) mice). DT-injected Rosa26-DT(Mlc2v-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-II(lo)CCR2(+) macrophages in DT-injected Rosa26-DT(Mlc2v-Cre) mice compared with diluent-treated Rosa26-DT(Mlc2v-Cre) mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DT(Mlc2v-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-DT(Mlc2v-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 acetylcholine, nicotine, and muscarine. To our knowledge, these findings reveal a previously unappreciated immunomodulatory role for the nonneuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury.

Study objective Recent evidence shows that subgroups of circulating B cells accumulate intravascularly in the naïve murine heart. However, the timing of their appearance and their organ specificity remain unclear. Methods and Results We performed a systematic analysis of B cells isolated from the myocardium and other organs, from mid development to early adulthood. We found that B cells are present in the developing heart already at E13.5. Myocardial associated B cells were mostly intravascular and in close association with the endothelium. Their phenotype changed rapidly. Through flow cytometry and proteogenomics analysis we found that heart associated B cells were initially almost exclusively CD11b+ and CD11b−CD21−CD23− but became mostly CD11b−CD21+CD23+ cells at 5 weeks of age. Importantly, comparative analysis of B cells isolated from perfused hearts, livers, lungs, spleen and peripheral blood at different time points showed that the B cells isolated from the heart were in equilibrium with both splenic/circulating B cells and B cells isolated from other organs, but they had specific features. This finding was confirmed through simultaneous 10X single cells sequencing of hashtag labelled B cells. Conclusions Taken together these observations highlight that myocardial associated B cells are part of a tightly regulated population of circulating B lymphocytes that exists in multiple organs and is in dynamic equilibrium with the spleen and peripheral blood B cells. Further work will be needed to identify the molecular mechanisms that lead to the accumulation of specific subsets of circulating B cells in peripheral organs and to investigate their functional significance.

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.

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. β-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.

Heart activity and long-term function are regulated by the sympathetic and parasympathetic branches of the nervous system. Parasympathetic neurons have received increased attention recently because acetylcholine (ACh) has been shown to play protective roles in heart disease. However, parasympathetic innervation is sparse in the heart, raising the question of how cholinergic signaling regulates cardiomyocytes. We hypothesized that non-neuronal secretion of ACh from cardiomyocytes plays a role in cholinergic regulation of cardiac activity. To test this possibility, we eliminated secretion of ACh exclusively from cardiomyocytes by targeting the vesicular acetylcholine transporter (VAChT). We find that lack of cardiomyocyte-secreted ACh disturbs the regulation of cardiac activity and causes cardiomyocyte remodeling. Mutant mice present normal hemodynamic parameters under nonstressful conditions; however, following exercise, their heart rate response is increased. Moreover, hearts from mutant mice present increased oxidative stress, altered calcium signaling, remodeling, and hypertrophy. Hence, without cardiomyocyte-derived ACh secretion, hearts from mutant mice show signs of imbalanced autonomic activity consistent with decreased cholinergic drive. These unexpected results suggest that cardiomyocyte-derived ACh is required for maintenance of cardiac homeostasis and regulates critical signaling pathways necessary to maintain normal heart activity. We propose that this non-neuronal source of ACh boosts parasympathetic cholinergic signaling to counterbalance sympathetic activity regulating multiple aspects of heart physiology.—Roy, A., Fields, W. C., Rocha-Resende, C., Resende, R. R., Guatimosim, S., Prado, V. F., Gros, R., Prado, M. A. M. Cardiomyocyte-secreted acetylcholine is required for maintenance of homeostasis in the heart.

High serum levels of aldosterone have been linked to the development of cardiac disease. In contrast, angiotensin (Ang)-(1-7) was extensively shown to possess cardioprotective effects, including the attenuation of cardiac dysfunction induced by excessive mineralocorticoid activation in vivo, suggesting possible interactions between these 2 molecules. Here, we investigated whether there is cross-talk between aldosterone and Ang-(1-7) and its functional consequences for calcium (Ca 2+ ) signaling in ventricular myocytes. Short-term effects of aldosterone on Ca 2+ transient were assessed in Fluo-4/AM-loaded myocytes. Confocal images showed that Ang-(1-7) had no effect on Ca 2+ transient parameters, whereas aldosterone increased the magnitude of the Ca 2+ transient. Quite unexpectedly, addition of Ang-(1-7) to aldosterone-treated myocytes further enhanced the amplitude of the Ca 2+ transient suggesting a synergistic effect of these molecules. Aldosterone action on Ca 2+ transient amplitude was mediated by protein kinase A, and was related to an increase in Ca 2+ current ( I Ca ) density. Both changes were not altered by Ang-(1-7). When cardiomyocytes were exposed to aldosterone, increased Ca 2+ spark rate was measured. Ang-(1-7) prevented this change. In addition, a NO synthase inhibitor restored the effect of aldosterone on Ca 2+ spark rate in Ang-(1-7)-treated myocytes and attenuated the synergistic effect of these 2 molecules on Ca 2+ transient. These results indicate that NO plays an important role in this cross-talk. Our results bring new perspectives in the understanding of how 2 prominent molecules with supposedly antagonist cardiac actions cross-talk to synergistically amplify Ca 2+ signals in cardiomyocytes.

It is well established that inositol 1,4,5-trisphosphate (IP3) dependent Ca(2+) signaling plays a crucial role in cardiomyocyte hypertrophy. However, it is not yet known whether nuclear IP3 represents a Ca(2+) mobilizing pathway involved in this process. The goal of the current work was to investigate the specific role of nuclear IP3 in cardiomyocyte hypertrophic response. In this work, we used an adenovirus construct that selectively buffers IP3 in the nuclear region of neonatal cardiomyocytes. We showed for the first time that nuclear IP3 mediates endothelin-1 (ET-1) induced hypertrophy. We also found that both calcineurin (Cn)/nuclear factor of activated T Cells (NFAT) and histone deacetylase-5 (HDAC5) pathways require nuclear IP3 to mediate pathological cardiomyocyte growth. Additionally, we found that nuclear IP3 buffering inhibited insulin-like growth factor-1 (IGF-1) induced hypertrophy and prevented reexpression of fetal gene program. Together, these results demonstrated that nuclear IP3 is an essential and a conserved signal for both pathological and physiological forms of cardiomyocyte hypertrophy.

The renin-angiotensin system (RAS) plays a key role inthe modulation of multiple functions in many organs. It has been demonstrated that the protective axis Ang-(1-7)/ACE2/Mas exerts anti-inflammatory effects in different disorders. Along this line, we have shown that Mas receptor deficiency exacerbates lipopolysaccharide(LPS)-induced cerebral and systemic inflammation in mice. In this study, we investigate the effects of LPS-induced shock in a mouse model for global increases in Mas receptor expression (UB8 mice). Mice were injected intraperitoneally (i.p.) with LPS, and experiments were performed 1h (RT-qPCR and ELISA) and 6 h (Echocardiography, NAG activity, and Lung histological analysis) after injection (except for the survival curve experiment). Cytokine gene expression was significantly attenuated in the left ventricle (LV) of UB8 vs. FVBN mice (p

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 Ca

RNA interfering (RNAi) is a biological process that operates in most eukaryotic cells in which small interfering RNA (siRNA) molecules inhibit gene expression. Recently, RNAi has been recognized as a therapeutic option to treat several diseases. However, the use of this gene silence technology has been limited, mainly because of the low efficiency of the different vectors in delivering significant amounts of siRNA to cells. In this context, CNTs have emerged as a novel vector to deliver siRNA for post transcriptional gene silencing purposes in vitro and in vivo due to their unique chemical and physical properties. This chapter is focused on the various strategies available and recent developments on the applications of Single Wall Carbon Nanotubes (SWCNTs) as a nanovector for siRNA delivery.

In order to better understand the relationship between the primary structure of TsHpt-I - a bradykinin-potentiating peptide (BPP) isolated from the venom of the yellow scorpion Tityus serrulatus, with a non-canonical Lys residue prior to the conservative Pro-Pro doublet - and its cardiovascular effects, a series of ladder peptides were synthesized using the C-terminal portion of TsHpt-I as a template. All synthetic peptides having the Pro-Pro doublet at their C-terminal were able to potentiate the hypotensive effect of bradykinin. Conversely, only those analogues having Lys residue could induce a transient hypotension when intravenously administrated in male rats, indicating that the positive charge located toward the radical of this amino acid residue is crucial for this cardiovascular effect. Differently from all known BPPs, TsHpt-I acts as an agonist of the B(2) receptor and does not inhibit angiotensin-converting enzyme. The capacity of this peptide to activate this subtype of kinin receptor, releasing NO, was also affected by the absence of Lys' side-chain positive charge. Moreover, this study has demonstrated that the minimization of the primary structure of TsHpt-I does not significantly alter the biological effects of this native peptide, which could be of interest for biotechnological purposes.

Alamandine is a new component of the renin-angiotesin system generated from angiotensin A or angiotensin-(1[[Unable to Display Character: –]]7). Its biological actions include vasodilation, and antihypertensive effects. In the heart, the molecular pathways activated by alamandine have not been characterized. Our goal was to investigate the signaling pathways activated by alamandine in ventricular myocytes isolated from both healthy and hypertensive models. Cardiomyocytes isolated from C57BL/6 mice and from rats that overexpress renin (TGRmRen) were treated with 100nmol/L alamandine. Intracellular nitric oxide (NO) and Ca 2+ levels were recorded in cells loaded with DAF-FM and Fluo4-AM, respectively. Protein phosphorylation was assessed by western-blot. In cardiomyocytes, alamandine induced an increase in phosphorylation of PDK1 (arbitrary units (a.u.): control=0,20±0.06 versus alamandine=0,42±0.08 n=6, p2+ transient amplitude. Ventricular myocytes treated with alamandine showed decreased GSK3β phosphorylation (a.u.: control=0,62±0.19 versus alamandine=0,50±0.25 n=5 p2 ): control=447±23 n=58; angiotensin II= 609±26 n=70; angiotensin II+alamandine= 491±31 n=45 p

Background Succinate is an intermediate of the citric acid cycle as well as an extracellular circulating molecule, whose receptor, G protein-coupled receptor-91 (GPR91), was recently identified and characterized in several tissues, including heart. Because some pathological conditions such as ischemia increase succinate blood levels, we investigated the role of this metabolite during a heart ischemic event, using human and rodent models. Results We found that succinate causes cardiac hypertrophy in a GPR91 dependent manner. GPR91 activation triggers the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), the expression of calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) and the translocation of histone deacetylase 5 (HDAC5) into the cytoplasm, which are hypertrophic-signaling events. Furthermore, we found that serum levels of succinate are increased in patients with cardiac hypertrophy associated with acute and chronic ischemic diseases. Conclusions These results show for the first time that succinate plays an important role in cardiomyocyte hypertrophy through GPR91 activation, and extend our understanding of how ischemia can induce hypertrophic cardiomyopathy. Electronic supplementary material The online version of this article (doi:10.1186/s12964-014-0078-2) contains supplementary material, which is available to authorized users.

Cholinergic control of the heart is exerted by two distinct branches; the autonomic component represented by the parasympathetic nervous system, and the recently described non-neuronal cardiomyocyte cholinergic machinery. Previous evidence has shown that reduced cholinergic function leads to deleterious effects on the myocardium. Yet, whether conditions of increased cholinergic signaling can offset the pathological remodeling induced by sympathetic hyperactivity, and its consequences for these two cholinergic axes are unknown. Here, we investigated two models of sympathetic hyperactivity: i) the chronic beta-adrenergic receptor stimulation evoked by isoproterenol (ISO), and ii) the alpha(2A)/alpha(2C)-drenergic receptor knockout (KO) mice that lack pre-synaptic adrenergic receptors. In both models, cholinergic signaling was increased by administration of the cholinesterase inhibitor, pyridostigmine. First, we observed that isoproterenol produces an autonomic imbalance characterized by increased sympathetic and reduced parasympathetic tone. Under this condition transcripts for cholinergic proteins were upregulated in ventricular myocytes, indicating that non-neuronal cholinergic machinery is activated during adrenergic overdrive. Pyridostigmine treatment prevented the effects of ISO on autonomic function and on the ventricular cholinergic machinery, and inhibited cardiac remodeling. alpha(2A)/alpha(2C)-KO mice presented reduced ventricular contraction when compared to wild-type mice, and this dysfunction was also reversed by cholinesterase inhibition. Thus, the cardiac parasympathetic system and non-neuronal cardiomyocyte cholinergic machinery are modulated in opposite directions under conditions of increased sympathetic drive or ACh availability. Moreover, our data support the idea that pyridostigmine by restoring ACh availability is beneficial in heart disease.

Recent work has provided compelling evidence that increased levels of acetylcholine (ACh) can be protective in heart failure, whereas reduced levels of ACh secretion can cause heart malfunction. Previous data show that cardiomyocytes themselves can actively secrete ACh, raising the question of whether this cardiomyocyte derived ACh may contribute to the protective effects of ACh in the heart. To address the functionality of this non-neuronal ACh machinery, we used cholinesterase inhibitors and a siRNA targeted to AChE (acetylcholinesterase) as a way to increase the availability of ACh secreted by cardiac cells. By using nitric oxide (NO) formation as a biological sensor for released ACh, we showed that cholinesterase inhibition increased NO levels in freshly isolated ventricular myocytes and that this effect was prevented by atropine, a muscarinic receptor antagonist, and by inhibition of ACh synthesis or vesicular storage. Functionally, cholinesterase inhibition prevented the hypertrophic effect as well as molecular changes and calcium transient alterations induced by adrenergic overstimulation in cardiomyocytes. Moreover, inhibition of ACh storage or atropine blunted the anti-hypertrophic action of cholinesterase inhibition. Altogether, our results show that cardiomyocytes possess functional cholinergic machinery that offsets deleterious effects of hyperadrenergic stimulation. In addition, we show that adrenergic stimulation upregulates expression levels of cholinergic components. We propose that this cardiomyocyte cholinergic signaling could amplify the protective effects of the parasympathetic nervous system in the heart and may counter-act or partially neutralize hypertrophic adrenergic effects.

International audience; Using a proteomic approach, a new structural family of peptides was put in evidence in the venom of the yellow scorpion Tityus serrulatus. Tityus serrulatus Hypotensins (TsHpt) are random-coiled linear peptides and have a similar bradykinin-potentiating peptide (BPP) amino acid signature. TsHpt-I (2.7kDa), the first member of this family, was able to potentiate the hypotensive effects of bradykinin (BK) in normotensive rats. Using the C-terminal of this peptide as a template, a synthetic analog peptide (TsHpt-I([17-25])) was designed to held the BK-potentiating effect. A relevant hypotensive effect, independent on BK, was also observed on both TsHpt (native and synthetic). To better evaluate this hypotensive effect, we examined the vasorelaxation of aortic rings from male Wistar rats and the peptides were able to induce endothelium-dependent vasorelaxation dependent on NO release. Both TsHpt could not inhibit ACE activity. These peptides appear to exert their anti-hypertensive effect through NO-dependent and ACE-independent mechanisms.

Our group has previously demonstrated that cardiomyocytes express ChAT (choline acetyltransferase), VAChT (vesicular acetylcholine transporter) and ChT-1 (choline high affinity transporter), proteins involved in the synthesis, release and reuptake of acetylcholine. Additionally, we have shown that this cardiomyocyte intrinsic cholinergic machinery has an important role in preventing deleterious effects of adrenergic signaling in vitro and in vivo. To address the role of this non-neuronal acetylcholine for heart function, a new mouse lineage was generated with deletion of VAChT (vesicular acetylcholine transporter) only in cardiac myocytes (cVAChT-ko). These mice are characterized by heart hypertrophy, calcium signaling dysfunction and increased reactive oxygen species levels. In order to better understand how impairment in acetylcholine released from cardiomyocytes could impact heart function, we subjected these mice to transverse aortic constriction (TAC) and evaluated left ventricular hypertrophy at 3 and 14 days after surgery. 3 days after TAC, cardiac hypertrophy was more prominent in cVAChT-ko mice than in control hearts (control: 11% versus cVAChT: 24%). However, 14 days after surgery, heart and left ventricular hypertrophy were similar between cVAChT-ko and control mice. Additionally, the lung/body ratio was significantly increased only in cVAChT-ko with constriction (control: 9% versus cVAChT-ko: 54%), suggesting that these mice present pulmonary edema, which is an indicative of heart failure. Moreover, TAC induced an increase in PLN/SERCA ratio only in cVAChT-ko mice. Thus our data show that cVAChT-ko mice is more prone to cardiac disease under stress conditions than control mice, indicating that cardiomyocyte intrinsic cholinergic machinery plays an important role in cardiac protection.FINANCIAL SUPPORT: CNPq, FAPEMIG, NIH (Fogarty International Award)