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Background: Immune cell–mediated inflammation is an essential process for mounting a repair response after myocardial infarction (MI). The sympathetic nervous system is known to regulate immune system function through β-adrenergic receptors (βARs); however, their role in regulating immune cell responses to acute cardiac injury is unknown. Methods: Wild-type (WT) mice were irradiated followed by isoform-specific βAR knockout (βARKO) or WT bone-marrow transplantation (BMT) and after full reconstitution underwent MI surgery. Survival was monitored over time, and alterations in immune cell infiltration after MI were examined through immunohistochemistry. Alterations in splenic function were identified through the investigation of altered adhesion receptor expression. Results: β 2 ARKO BMT mice displayed 100% mortality resulting from cardiac rupture within 12 days after MI compared with ≈20% mortality in WT BMT mice. β 2 ARKO BMT mice displayed severely reduced post-MI cardiac infiltration of leukocytes with reciprocally enhanced splenic retention of the same immune cell populations. Splenic retention of the leukocytes was associated with an increase in vascular cell adhesion molecule-1 expression, which itself was regulated via β-arrestin–dependent β 2 AR signaling. Furthermore, vascular cell adhesion molecule-1 expression in both mouse and human macrophages was sensitive to β 2 AR activity, and spleens from human tissue donors treated with β-blocker showed enhanced vascular cell adhesion molecule-1 expression. The impairments in splenic retention and cardiac infiltration of leukocytes after MI were restored to WT levels via lentiviral-mediated re-expression of β 2 AR in β 2 ARKO bone marrow before transplantation, which also resulted in post-MI survival rates comparable to those in WT BMT mice. Conclusions: Immune cell–expressed β 2 AR plays an essential role in regulating the early inflammatory repair response to acute myocardial injury by facilitating cardiac leukocyte infiltration.

Background We have recently found that 3 repeated doses (12×10 6 each) of c‐kit POS cardiac progenitor cells ( CPC s) were markedly more effective than a single dose of 12×10 6 cells in alleviating postinfarction left ventricular dysfunction and remodeling. However, since the single‐dose group received only one third of the total number of CPC s given to the multiple‐dose group, it is unknown whether the superior therapeutic efficacy was caused by repeated treatments per se or by administration of a higher total number of CPC s. This issue has major clinical implications because multiple cell injections in patients pose significant challenges, which would be obviated by using 1 large injection. Accordingly, we determined whether the beneficial effects of 3 repeated CPC doses can be recapitulated by 1 large dose containing the same total number of cells. Methods and Results Rats with a 30‐day‐old myocardial infarction received 3 echo‐guided intraventricular infusions, 35 days apart, of vehicle‐vehicle‐vehicle, 36×10 6 CPC s‐vehicle‐vehicle, or 3 equal doses of 12×10 6 CPC s. Infusion of a single, large dose of CPC s (36×10 6 cells) produced an initial improvement in left ventricular function, but no further improvement was observed after the second and third infusions (both vehicle). In contrast, each of the 3 doses of CPC s (12×10 6 ) caused a progressive improvement in left ventricular function, the cumulative magnitude of which was greater than with a single dose. Unlike the single dose, repeated doses reduced collagen content and immune cell infiltration. Conclusions Three repeated doses of CPC s are superior to 1 dose even though the total number of cells infused is the same, possibly because of greater antifibrotic and anti‐inflammatory actions.

Although, autophagy is an essential cellular salvage process to maintain cellular homeostasis, pathological autophagy can lead to cardiac abnormalities and ultimately heart failure. Therefore, a tight regulation on autophagic process would be important to treat chronic heart failure. Previously, we have shown that IL-10 strongly improved cardiac function in chronic heart failure models, but the role of IL-10 in regulation of pathological autophagy is not yet investigated. We tested the hypothesis that IL-10 inhibits angiotensin II-induced pathological autophagy and thus improved cardiac function. Pathological autophagy was induced in wild type (WT) and IL10-knockout mice by angiotensin II infusion. Ang II-induced left ventricular dysfunction and hypertrophic remodeling were accentuated in IL-10 KO mice compared to WT mice. IL-10 KO mice showed exaggerated autophagy with reduced AKT phosphorylation. In neonatal rat ventricular cardiomyocytes, Ang II activated beclin1 and LC3 levels and inhibited AKT/mTORC1 and AKT-Bcl2 signaling. IL-10 inhibited Ang II-induced autophagic marker proteins. Additionally, IL-10 restored Ang II effects on AKT/mTORC1 and AKT-Bcl2 signaling. Both pharmacological/molecular inhibition of AKT via PI3K inhibitor (LY290002) or Akt siRNA, attenuated IL-10 effects on the Ang II-induced pathological autophagy, confirming that IL-10 mediated regulation of pathological autophagy is AKT dependent. Similar results were observed with mTORC1 inhibitor rapamycin. Chloroquine (a lysosome inhibitor) strongly inhibits Ang II-induced autophagic flux. However, chloroquine did not affect IL-10 effects on autophagic flux, suggesting that IL-10 inhibits stress-induced pathological autophagy. Finally, as physical interaction of Bcl2 with beclin 1 is important to inhibit autophagy and IL-10 is strong activator of Bcl2, we performed immunoprecipitation experiment. Immunoprecipitation data suggested that Ang II disrupt the physical interaction of beclin 1 with Bcl2 and IL-10 reestablished this physical interaction to reduce autophagy. Our data give a novel role of IL-10 in regulation of pathological autophagy and thus can act as a potential therapeutic molecule in treatment of chronic heart disease.

MicroRNAs are known to be dysregulated in the ischemic heart disease and have emerged as potential therapeutic targets for treatment of myocardial infarction (MI). Recently MicroRNA-375 has been shown to be up-regulated in humans with MI. In this study, we assessed whether inhibition of the miR-375 using an i.v.-delivered locked nucleic acid (LNA)-modified anti-miR (LNA-antimiR-375) can provide therapeutic benefit in mice with pre-existing pathological cardiac remodeling and dysfunction due to myocardial infarction (MI).After the induction of acute myocardial infarction, mice were treated with either control or LNA based miR-375 inhibitor, and inflammatory response, cardiomyocyte apoptosis and LV functional and structural remodeling changes were evaluated. Anti-miR-375 therapy significantly suppressed infiltration of inflammatory cells, expression of proinflammatory cytokines in the myocardium and cardiomyocyte apoptosis. These changes were associated with miR-375 mediated activation of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) and downstream AKT phosphorylation on Thr-308. LNA anti-miR-375 therapy significantly improved LV functions, reduced infarct size, and attenuated infarct wall thinning. Moreover, LNA based miR-375 therapy significantly increased capillary density in the infarcted myocardium. Further, our in vitro studies demonstrated that miR-375 negatively regulates the expression of PDK-1 by directly targeting the 3’UTR of the PDK-1 transcript. Taken together, our studies demonstrate that anti miR-375 therapy suppresses inflammatory response, cardiomyocyte death and contributes to improved LV function and enhanced angiogenesis via activation of PDK-1/AKT signaling.

β-adrenergic receptors (βAR) are critical regulators of cardiac function normally and during (HF). The importance of βAR on cardiomyocyte contractility and survival is well defined however, following myocardial infarction (MI), inflammatory responses occur, which are critical for healing and scar formation. Catecholamines acting through βAR, particularly the β2AR subtype, are known to modulate immune responses, however, the influence of β2AR in regulating the inflammatory response following MI is unknown. To investigate the contribution of β2AR on immune cells following myocardial infarction (MI), wild-type (WT) mice were irradiated and then received β2ARKO or WT control BM transplants to create immune cell specific knockout (KO) animals. Following bone marrow reconstitution, mice were subjected to MI and cardiac function and survival were monitored. Cardiac function, as assessed by echocardiography, did not differ between WT and β2ARKO chimeric mice. However, mice lacking β2ARKO in their BM resulted in 100% mortality from cardiac rupture within two weeks of receiving MI in contrast to their WT counterparts that had ~20% death. Masson trichrome staining demonstrated infarct expansion in β2ARKO chimeric mice occurred more rapidly than their WT counterparts. Flow cytometric analysis showed decreased mobilization of granulocytes from bone marrow in β2ARKO mice. Additionally, β2ARKO chimeric mice reductions in infiltrating monocyte/macrophage, neutrophil and mast cell populations in the heart with no change in total cell infiltration suggesting a disruption in the ratio of infiltrating immune cells. Alterations in chemokine receptor levels, particularly CCR2, on BM resulted in decreased cellular migration. These results demonstrate the critical role of β2AR in mounting an immune response and promoting healing following MI.

Myocardial ischemic injury is characterized by an extensive loss of myocytes due to apoptosis/necrosis followed by an immune/inflammatory response and reestablishment of tissue integrity by a fibrotic scar. Prevention of rupture due to scar formation however, compromises contractility and reduces cardiac function. As bone marrow derived stem/precursor cells (BMSCs) have the ability to trans-differentiate and aid in the regeneration of injured myocardium, we studied BMSCs and their response to inflammation. BM cell mobilization, egress from the BM and recruitment to the site of injury can be regulated by signals through G protein-coupled receptors (GPCRs). β-arrestins are known for their GPCR signaling termination and scaffolding functions. Here, we explored the potential role for β-arrestins expressed in total BM in mediation of the initiation and progression of inflammation. We investigated the role β-arrestin1 (βArr1) and β-arrestin2 (βArr2) with respect to modulation of regenerative competence of BMSCs and inflammatory cells and their contribution to cardiac repair following ischemic injury. We carried out BM transplants to determine whether βArrs may be involved in cardiac repair. WT mice were irradiated and received BM transplants from WT, βArr1 or βArr2 KO donor mice. Subsequent to BM reconstitution, mice underwent MI and their recovery and progress were followed. Interestingly, βArr1 and βArr2 KO chimeras had inferior outcomes than mice receiving WT BM. This included significantly decreased post-MI survival with βArr2 KO BM and both βArr chimeras had significantly lower cardiac function post-MI compared to WT BM recipients. Histology shows that both chimeras had larger infarcts, accelerated rate of hypertrophy, quicker induction of fibrosis, exacerbated cell infiltration and increased number of apoptotic cells. At 3d post-MI immune cell intrusion of βArr2KO chimeric hearts was highest and the blood plasma content of TNFα and IL-6 was increased. We conclude that βArrs play a novel role downstream of GPCR desensitization in cardiac progenitor/inflammatory cells in the BM and are critical in the heart’s response to ischemic injury via cardiac repair and regeneration.

Chronic heart failure after myocardial injury (MI) is characterized by an extensive loss of myocytes due to considerable cell death. Bone marrow derived stem cells (BMSCs) can transdifferentiate and show potential for regenerating the myocardium after MI. Stem cell mobilization, egress from the bone marrow and recruitment to the site of injury can be regulated by signals through G protein coupled receptors (GPCRs). βArrestins have signalling and scaffolding functions and act as downstream regulators of GPCR desensitization and endocytosis. We explored the potential role for βArrestins in cardiac precursor cell function, concentrating on BMSCs. Using knockout (KO) mice, we investigated the role βArrestin1 (βArr1) and βArrestin2 (βArr2), their modulation of regenerative competence of BMSCs and their contribution to cardiac repair after ischemic injury. in vitro, we observed that BM derived cells devoid of either βArr1 or βArr2 are slower to proliferate, colonize and migrate, compared to wild type (WT) BM cells. We also observed elevated cell death in βArr2 deficient cells following oxidative stress. Additionally, the number of cKit+ stem cells, thought to be potential cardiac precursor cells, was significantly lower in the BM and blood of βArr KO vs WT. Similarly, BM and blood of the chimeras contained fewer and less viable cardiac stem/precursor cells pre and post MI, compared to WT transplanted controls. In our in vivo study, we carried out BM transplants to determine whether the βArrs may be involved in cardiac repair. WT mice were irradiated and received BM transplants from WT, βArr1 KO or βArr2 KO mice. Following BM reconstitution, mice underwent MI and their recovery was monitored. Interestingly, chimeric mice with βArr1 and βArr2 KO BM had significantly inferior outcomes, including significantly decreased post MI survival with βArr2 KO BM and both βArr chimeras had significantly lower cardiac function post MI than mice receiving WT BM. Histology revealed that both chimeras developed larger infarcts and hypertrophy at an faster rate. We conclude that βArrs play a novel role downstream of GPCR desensitization in cardiac progenitor cells in BM and appear to be critically involved in the heart’s response to ischemic injury via cardiac repair and regeneration.

Development of chronic heart failure (HF) syndrome following myocardial injury (MI) is characterized by an extensive loss of myocytes due to substantial apoptosis and necrosis. As bone marrow derived stem cells (BMSCs) are capable of transdifferentiating, they also show potential for regenerating the myocardium after infarction. Stem cell mobilization, egress from the bone marrow and homing to the site of injury can be regulated by signals through G protein-coupled receptors (GPCRs). β-arrestins are known for their signalling and scaffolding functions and as downstream regulators of GPCR desensitization and endocytosis in particular. We have begun to investigate whether β-arrestins play any role in cardiac precursor cell function concentrating on the properties of BMSCs. Using knockout (KO) mice, we investigated the role β-arrestin1 (βArr1) and β-arrestin2 (βArr2) with respect to modulation of regenerative competence of BMSCs and their contribution to cardiac repair following ischemic injury. First, BM-derived cells devoid of either βArr1 or βArr2 were found to proliferate and colonize in a significantly deficient manner compared to BM cells isolated from wild-type (WT) mice. In addition, the number of c-kit positive cardiac stem cells (as a % of total BM) were significantly lower in the βArr KO mice compared to WT. We carried out BM transplant studies to begin to determine whether the βArrs may play a role in cardiac repair. In our study, WT mice were irradiated and then received BM transplants from either WT donors as a control or BM from βArr1 or βArr2 KO mice. Subsequent to BM reconstitution, mice underwent a myocardial infarction (MI) and their condition was followed. Interestingly, chimeric mice with βArr1 and βArr2 KO BM had significantly poorer outcomes than mice receiving WT BM. This included significantly decreased post-MI survival with βArr2 KO BM and both βArr chimeras had significantly lower cardiac function post-MI compared to mice receiving WT BM. Additionally, our analyses of the BM cells and very small embryonic-like stem cells (VSELs) circulating in peripheral blood (PB), indicate that βArr-KO BM and PB contain fewer and less viable cardiac stem/precursor cells compared to WT transplanted controls.