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Repeated doses of c-kit(+) cardiac progenitor cells (CPCs) are superior to a single dose in improving LV function in rats with old myocardial infarction (MI). However, this concept needs testing in different species to determine whether it is generalizable. We used a new murine model of chronic ischemic cardiomyopathy whose unique feature is that cell therapy was started late (3 months) after MI. Mice received three echo-guided intraventricular infusions, 5 weeks apart, of vehicle, CPCs × 1, or CPCs × 3. Echocardiography demonstrated that the single-dose group exhibited improved LV ejection fraction (EF) after the 1(st) infusion (CPCs), but not after the 2(nd) and 3(rd) (vehicle). In contrast, in the multiple-dose group LVEF continued to improve, so that the final value was greater than in vehicle or single-dose groups (P

Mounting evidence shows that cell therapy provides therapeutic benefits in experimental and clinical settings of chronic heart failure. However, direct cardiac delivery of cells via transendocardial injection is logistically complex, expensive, entails risks, and is not amenable to multiple dosing. Intravenous administration would be a more convenient and clinically applicable route for cell therapy. Thus, we determined whether intravenous infusion of three widely used cell types improves left ventricular (LV) function and structure and compared their efficacy. Rats with a 30-day-old myocardial infarction (MI) received intravenous infusion of vehicle (PBS) or 1 of 3 types of cells: bone marrow mesenchymal stromal cells (MSCs), cardiac mesenchymal cells (CMCs), and c-kit-positive cardiac cells (CPCs), at a dose of 12x106 cells. Rats were followed for 35 days after treatment to determine LV functional status by serial echocardiography and hemodynamic studies. Blood samples were collected for Hemavet analysis to determine inflammatory cell profile. LV ejection fraction (EF) dropped ≥20 points in all hearts at 30-day after MI and deteriorated further at 35-day follow-up in the vehicle-treated group. In contrast, deterioration of EF was halted in rats that received MSCs and attenuated in those that received CMCs or CPCs. None of the 3 types of cells significantly altered scar size, myocardial content of collagen or CD45-positive cells, or Hemavet profile. This study demonstrates that a single intravenous administration of 3 types of cells in rats with chronic ischemic cardiomyopathy is effective in attenuating the progressive deterioration in LV function. The extent of LV functional improvement was greatest with CPCs, intermediate with CMCs, and least with MSCs.

Intramyocardial injection of synthetic microRNAs (miRs) has recently been reported to be beneficial after myocardial infarction (MI). We conducted a randomized blinded study to evaluate the efficacy and reproducibility of this strategy in a mouse model of reperfused MI using rigorous methodology. Mice undergoing a 60-min coronary occlusion followed by reperfusion were randomly assigned to control miR, hsa-miR-199a-3p, hsa-miR-149-3p, or hsa-miR-149-5p mimic treatment. Intramyocardial injections of miRs were performed in the border zone right after reperfusion. At 8 weeks after MI, there were no significant differences in ejection fraction (EF) among groups (EF = 27.1 ± 0.4% in control group [n = 6] and 25.9 ± 0.5%, 26.0 ± 0.8%, and 26.6 ± 0.6% in hsa-miR-199a-3p, hsa-miR-149-3p, or hsa-miR-149-5p groups, respectively [n = 9 each]). Net change (delta) in EF at 8 weeks compared with day 3 after MI was − 4.1% in control and − 3.2%, − 2.4%, and − 0.4% in the miR-treated groups (P = NS). Assessment of cardiac function by hemodynamic studies (a method independent of echocardiography) confirmed that there was no difference in left ventricular systolic or diastolic function among groups. Consistent with the functional data, histological analysis showed no difference in scar size, cardiomyocyte area, capillary density, collagen content, or apoptosis among groups. In conclusion, this randomized, blinded study demonstrates that intramyocardial injection of a single dose of synthetic hsa-miR-199a-3p, hsa-miR-149-3p, or hsa-miR-149-5p mimic does not improve cardiac function or remodeling in a murine model of reperfused MI. The strategy of using synthetic miR mimics for cardiac repair after MI needs to be evaluated with rigorous preclinical studies before its potential clinical translation.

Although cell therapy-mediated cardiac repair offers promise for treatment/management of heart failure, lack of fundamental understanding of how cell therapy works limits its translational potential. In particular, whether reparative cells from failing hearts differ from cells derived from nonfailing hearts remains unexplored. Here, we assessed differences between cardiac mesenchymal cells (CMC) derived from failing (HF) versus nonfailing (Sham) hearts and whether the source of donor cells (i.e., from HF vs. Sham) limits reparative capacity, particularly when administered late after infarction. To determine the impact of the donor source of CMCs, we characterized the transcriptional profile of CMCs isolated from sham (Sham-CMC) and failing (HF-CMC) hearts. RNA-seq analysis revealed unique transcriptional signatures in Sham-CMC and HF-CMC, suggesting that the donor source impacts CMC. To determine whether the donor source affects reparative potential, C57BL6/J female mice were subjected to 60 min of regional myocardial ischemia and then reperfused for 35 days. In a randomized, controlled, and blinded fashion, vehicle, HF-CMC, or Sham-CMC were injected into the lumen of the left ventricle at 35 days post-MI. An additional 5 weeks later, cardiac function was assessed by echocardiography, which indicated that delayed administration of Sham-CMC and HF-CMC attenuated ventricular dilation. We also determined whether Sham-CMC and HF-CMC treatments affected ventricular histopathology. Our data indicate that the donor source (nonfailing vs. failing hearts) affects certain aspects of CMC, and these insights may have implications for future studies. Our data indicate that delayed administration of CMC limits ventricular dilation and that the source of CMC may influence their reparative actions. NEW & NOTEWORTHY Most preclinical studies have used only cells from healthy, nonfailing hearts. Whether donor condition (i.e., heart failure) impacts cells used for cell therapy is not known. We directly tested whether donor condition impacted the reparative effects of cardiac mesenchymal cells in a chronic model of myocardial infarction. Although cells from failing hearts differed in multiple aspects, they retained the potential to limit ventricular remodeling.

Rationale The beta-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to proteins is a pro-adaptive response to cellular insults. To this end, increased protein O-GlcNAcylation improves short-term survival of cardiomyocytes subjected to acute injury. This observation has been repeated by multiple groups and in multiple models; however, whether increased protein O-GlcNAcylation plays a beneficial role in more chronic settings remains an open question. Objective Here, we queried whether increasing levels of cardiac protein O-GlcNAcylation would be beneficial during infarct-induced heart failure. Methods and results To achieve increased protein O-GlcNAcylation, we targeted Oga, the gene responsible for removing O-GlcNAc from proteins. Here, we generated mice with cardiomyocyte-restricted, tamoxifen-inducible haploinsufficient Oga gene. In the absence of infarction, we observed a slight reduction in ejection fraction in Oga deficient mice. Overall, Oga reduction had no major impact on ventricular function. In additional cohorts, mice of both sexes and both genotypes were subjected to infarct-induced heart failure and followed for up to four weeks, during which time cardiac function was assessed via echocardiography. Contrary to our prediction, the Oga deficient mice exhibited exacerbated—not improved—cardiac function at one week following infarction. When the observation was extended to 4 wk post-MI, this acute exacerbation was lost. Conclusions The present findings, coupled with our previous work, suggest that altering the ability of cardiomyocytes to either add or remove O-GlcNAc modifications to proteins exacerbates early infarct-induced heart failure. We speculate that more nuanced approaches to regulating O-GlcNAcylation are needed to understand its role—and, in particular, the possibility of cycling, in the pathophysiology of the failing heart.

Background The authors previously reported that the c-kit–positive (c-kitPOS) cells isolated from slowly adhering (SA) but not from rapidly adhering (RA) fractions of cardiac mesenchymal cells (CMCs) are effective in preserving left ventricular (LV) function after myocardial infarction (MI). Objectives This study evaluated whether adherence to plastic alone, without c-kit sorting, was sufficient to isolate reparative CMCs. Methods RA and SA CMCs were isolated from mouse hearts, expanded in vitro, characterized, and evaluated for therapeutic efficacy in mice subjected to MI. Results Morphological and phenotypic analysis revealed that murine RA and SA CMCs are indistinguishable; nevertheless, transcriptome analysis showed that they possess fundamentally different gene expression profiles related to factors that regulate post-MI LV remodeling and repair. A similar population of SA CMCs was isolated from porcine endomyocardial biopsy samples. In mice given CMCs 2 days after MI, LV ejection fraction 28 days later was significantly increased in the SA CMC group (31.2 ± 1.0% vs. 24.7 ± 2.2% in vehicle-treated mice; p < 0.05) but not in the RA CMC group (24.1 ± 1.2%). Histological analysis showed reduced collagen deposition in the noninfarcted region in mice given SA CMCs (7.6 ± 1.5% vs. 14.5 ± 2.8% in vehicle-treated mice; p < 0.05) but not RA CMCs (11.7 ± 1.7%), which was associated with reduced infiltration of inflammatory cells (14.1 ± 1.6% vs. 21.3 ± 1.5% of total cells in vehicle and 19.3 ± 1.8% in RA CMCs; p < 0.05). Engraftment of SA CMCs was negligible, which implies a paracrine mechanism of action. Conclusions We identified a novel population of c-kit–negative reparative cardiac cells (SA CMCs) that can be isolated with a simple method based on adherence to plastic. SA CMCs exhibited robust reparative properties and offered numerous advantages, appearing to be more suitable than c-kitPOS cardiac progenitor cells for widespread clinical therapeutic application.

Several post-translational modifications figure prominently in ventricular remodeling. The beta-O-linkage of N-acetylglucosamine (O-GlcNAc) to proteins has emerged as an important signal in the cardiovascular system. Although there are limited insights about the regulation of the biosynthetic pathway that gives rise to the O-GlcNAc post-translational modification, much remains to be elucidated regarding the enzymes, such as O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which regulate the presence/absence of O-GlcNAcylation. Recently, we showed that the transcription factor, E2F1, could negatively regulate OGT and OGA expression in vitro. The present study sought to determine whether E2f1 deletion would improve post-infarct ventricular function by de-repressing expression of OGT and OGA. Male and female mice were subjected to non-reperfused myocardial infarction (MI) and followed for 1 or 4 week. MI significantly increased E2F1 expression. Deletion of E2f1 alone was not sufficient to alter OGT or OGA expression in a naïve setting. Cardiac dysfunction was significantly attenuated at 1-week post-MI in E2f1-ablated mice. During chronic heart failure, E2f1 deletion also attenuated cardiac dysfunction. Despite the improvement in function, OGT and OGA expression was not normalized and protein O-GlcNAcyltion was not changed at 1-week post-MI. OGA expression was significantly upregulated at 4-week post-MI but overall protein O-GlcNAcylation was not changed. As an alternative explanation, we also performed guided transcriptional profiling of predicted targets of E2F1, which indicated potential differences in cardiac metabolism, angiogenesis, and apoptosis. E2f1 ablation increased heart size and preserved remote zone capillary density at 1-week post-MI. During chronic heart failure, cardiomyocytes in the remote zone of E2f1-deleted hearts were larger than wildtype. These data indicate that, overall, E2f1 exerts a deleterious effect on ventricular remodeling. Thus, E2f1 deletion improves ventricular remodeling with limited impact on enzymes regulating O-GlcNAcylation.

Using a murine model of chronic ischemic cardiomyopathy caused by an old myocardial infarction (MI), we have previously found that three doses of 1 × 106 c-kit positive cardiac cells (CPCs) are more effective than a single dose of 1 × 106 cells. The goal of this study was to determine whether the beneficial effects of three doses of CPCs (1 × 106 cells each) can be fully replicated by a single combined dose of 3 × 106 CPCs. Mice underwent a 60-min coronary occlusion, after 90 days of reperfusion, they received three echo-guided intraventricular infusions at 5-week intervals: (1) vehicle × 3, (2) one combined dose of CPCs (3 × 106) and vehicle × 2, or (3) three doses of CPCs (1 × 106 each). In the combined-dose group, left ventricular ejection fraction (LVEF) improved after the 1st CPC infusion, but not after the 2nd and 3rd (vehicle) infusions. In contrast, in the multiple-dose group, LVEF increased after each CPC infusion, at the final echo, LVEF averaged 35.2 ± 0.6% (p &lt, 0.001 vs. the vehicle group, 27.3 ± 0.2%). At the end of the study, the total cumulative change in EF from pretreatment values was numerically greater in the multiple-dose group (6.6 ± 0.6%) than in the combined-dose group (4.8 ± 0.8%), although the difference was not statistically significant (p = 0.08). Hemodynamic studies showed that several parameters of LV function in the multiple-dose group were numerically greater than in the combined-dose group (p = 0.08 for the difference in LVEF). Compared with vehicle, cardiomyocyte cross-sectional area was reduced only in the multiple-dose group (−32.7%, 182.6 ± 15.1 µm2 vs. 271.5 ± 27.2 µm2, p &lt, 0.05, in the risk region and −28.5%, 148.5 ± 12.1 µm2 vs. 207.6 ± 20.5 µm2, p &lt, 0.05, in the noninfarcted region). LV weight/body weight ratio and LV weight/tibia length ratios were significantly reduced in both cell treated groups vs. the vehicle group, indicating the attenuation of LV hypertrophy, however, the lung weight/body weight ratio was significantly reduced only in the multiple-dose group, suggesting decreased pulmonary congestion. Taken together, these results indicate that in mice with chronic ischemic cardiomyopathy, the beneficial effects of three doses of CPCs on LV function and hypertrophy cannot be fully replicated with a single dose, notwithstanding the fact that the total number of cells delivered with one or three doses is the same. Thus, it is the multiplicity of doses, and not the total number of cells, that accounts for the superiority of the repeated-dose paradigm. This study supports the idea that the efficacy of cell therapy in heart failure can be augmented by repeated administrations.

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.

Preclinical investigations support the concept that donor cells more oriented towards a cardiovascular phenotype favor repair. In light of this philosophy, we previously identified HDAC1 as a mediator of cardiac mesenchymal cell (CMC) cardiomyogenic lineage commitment and paracrine signaling potency in vitro—suggesting HDAC1 as a potential therapeutically exploitable target to enhance CMC cardiac reparative capacity. In the current study, we examined the effects of pharmacologic HDAC1 inhibition, using the benzamide class 1 isoform-selective HDAC inhibitor entinostat (MS-275), on CMC cardiomyogenic lineage commitment and CMC-mediated myocardial repair in vivo. Human CMCs pre-treated with entinostat or DMSO diluent control were delivered intramyocardially in an athymic nude rat model of chronic ischemic cardiomyopathy 30 days after a reperfused myocardial infarction. Indices of cardiac function were assessed by echocardiography and left ventricular (LV) Millar conductance catheterization 35 days after treatment. Compared with naive CMCs, entinostat-treated CMCs exhibited heightened capacity for myocyte-like differentiation in vitro and superior ability to attenuate LV remodeling and systolic dysfunction in vivo. The improvement in CMC therapeutic efficacy observed with entinostat pre-treatment was not associated with enhanced donor cell engraftment, cardiomyogenesis, or vasculogenesis, but instead with more efficient inhibition of myocardial fibrosis and greater increase in myocyte size. These results suggest that HDAC inhibition enhances the reparative capacity of CMCs, likely via a paracrine mechanism that improves ventricular compliance and contraction and augments myocyte growth and function.

Background 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 of autophagic process would be important to treat chronic heart failure. Previously, we have shown that IL-10 strongly inhibited pressure overload-induced hypertrophy and heart failure, but role of IL-10 in regulation of pathological autophagy is unknown. Here we tested the hypothesis that IL-10 inhibits angiotensin II-induced pathological autophagy and this process, in part, leads to improve cardiac function. Methods and results Chronic Ang II strongly induced mortality, cardiac dysfunction in IL-10 Knockout mice. IL-10 deletion exaggerated pathological autophagy in response to Ang II treatment. In isolated cardiac myocytes, IL-10 attenuated Ang II-induced pathological autophagy and activated Akt/mTORC1 signaling. Pharmacological or molecular inhibition of Akt and mTORC1 signaling attenuated IL-10 effects on Ang II-induced pathological autophagy. Furthermore, lysosomal inhibition in autophagic flux experiments further confirmed that IL-10 inhibits pathological autophagy via mTORC1 signaling. Conclusion Our data demonstrate a novel role of IL-10 in regulation of pathological autophagy; thus can act as a potential therapeutic molecule for treatment of chronic heart disease.

Background: MicroRNA (miR) dysregulation in the myocardium has been implicated in cardiac remodeling after injury or stress.Objectives: The aim of this study was to explore the role of miR ...

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.

We have recently demonstrated that repeated administrations of c-kitPOS cardiac progenitor cells (CPCs) have cumulative beneficial effects in rats with old myocardial infarction (MI), resulting in markedly greater improvement in left ventricular (LV) function compared with a single administration. To determine whether this paradigm applies to other species and cell types, mice with a 3-week-old MI received one or three doses of cardiac mesenchymal cells (CMCs), a novel cell type that we have recently described. CMCs or vehicle were infused percutaneously into the LV cavity, 14 days apart. Compared with vehicle-treated mice, the single-dose group exhibited improved LV ejection fraction (EF) after the 1st infusion (consisting of CMCs) but not after the 2nd and 3rd (vehicle). In contrast, in the multiple-dose group, LV EF improved after each CMC infusion, so that at the end of the study, LV EF averaged 35.5 ± 0.7% vs. 32.7 ± 0.6% in the single-dose group (P

The myocardial response to pressure overload involves coordination of multiple transcriptional, posttranscriptional, and metabolic cues. The previous studies show that one such metabolic cue, O-GlcNAc, is elevated in the pressure-overloaded heart, and the increase in O-GlcNAcylation is required for cardiomyocyte hypertrophy in vitro. Yet, it is not clear whether and how O-GlcNAcylation participates in the hypertrophic response in vivo. Here, we addressed this question using patient samples and a preclinical model of heart failure. Protein O-GlcNAcylation levels were increased in myocardial tissue from heart failure patients compared with normal patients. To test the role of OGT in the heart, we subjected cardiomyocyte-specific, inducibly deficient Ogt (i-cmOgt -/-) mice and Ogt competent littermate wild-type (WT) mice to transverse aortic constriction. Deletion of cardiomyocyte Ogt significantly decreased O-GlcNAcylation and exacerbated ventricular dysfunction, without producing widespread changes in metabolic transcripts. Although some changes in hypertrophic and fibrotic signaling were noted, there were no histological differences in hypertrophy or fibrosis. We next determined whether significant differences were present in i-cmOgt -/- cardiomyocytes from surgically naive mice. Interestingly, markers of cardiomyocyte dedifferentiation were elevated in Ogt-deficient cardiomyocytes. Although no significant differences in cardiac dysfunction were apparent after recombination, it is possible that such changes in dedifferentiation markers could reflect a larger phenotypic shift within the Ogt-deficient cardiomyocytes. We conclude that cardiomyocyte Ogt is not required for cardiomyocyte hypertrophy in vivo; however, loss of Ogt may exert subtle phenotypic differences in cardiomyocytes that sensitize the heart to pressure overload-induced ventricular dysfunction.

Direct intercellular communication mediated by gap junctions (GJs) is a hallmark of normal cell and tissue physiology. In addition, GJs significantly contribute to physical cell-cell adhesion. Clearly, these cellular functions require precise modulation. Typically, GJs represent arrays of hundreds to thousands of densely packed channels, each one assembled from two half-channels (connexons), that dock head-on in the extracellular space to form the channel arrays that link neighboring cells together. Interestingly, docked GJ channels cannot be separated into connexons under physiological conditions, posing potential challenges to GJ channel renewal and physical cell-cell separation. We described previously that cells continuously-and effectively after treatment with natural inflammatory mediators-internalize their GJs in an endo-/exocytosis process that utilizes clathrin-mediated endocytosis components, thus enabling these critical cellular functions. GJ internalization generates characteristic cytoplasmic double-membrane vesicles, described and termed earlier annular GJs (AGJs) or connexosomes. Here, using expression of the major fluorescent-tagged GJ protein, connexin 43 (Cx43-GFP/YFP/mApple) in HeLa cells, analysis of endogenously expressed Cx43, ultrastructural analyses, confocal colocalization microscopy, pharmacological and molecular biological RNAi approaches depleting cells of key-autophagic proteins, we provide compelling evidence that GJs, following internalization, are degraded by autophagy. The ubiquitin-binding protein p62/sequestosome 1 was identified in targeting internalized GJs to autophagic degradation. While previous studies identified proteasomal and endo-/lysosomal pathways in Cx43 and GJ degradation, our study provides novel molecular and mechanistic insights into an alternative GJ degradation pathway. Its recent link to health and disease lends additional importance to this GJ degradation mechanism and to autophagy in general.

Intercellular communication mediated by exosomes, nano-sized extracellular vesicles, is crucial for preserving vascular integrity and in the development of cardiovascular and other diseases. As natural carriers of signal molecules, exosomes released from sources such as blood cells, endothelial cells, immune cells, smooth muscle cells, etc., can modify a multitude of cellular bioactivities. They do so by shuttling lipids, proteins, and nucleic acids between donor and recipient cells while circulating in body fluids and in the extracellular space. A recent surge of interest in the field of exosomal biology is in part due to the recognition that the molecules they carry can act as facilitators of both pathogenesis but can also initiate protective and rescue signaling. This mini-review describes current knowledge on exosome function in health and disease including cardiovascular disease.

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.

The continuous increase of average human life span is yielding a progressively older population pool. With the aging population and increasingly sedentary lifestyle, chronic cardiovascular diseases are approaching epidemic proportions in nearly all developed countries. Decline in cardiovascular performance is counteracted by the interaction of the sympathetic (adrenergic) nervous system (ANS) and the parasympathetic system. Acutely, the elevated activity of the adrenergic system will swiftly reestablish cardiac function and return to steady levels. However, a chronic manifestation of altered cardiac function is followed by prolonged and compensatory ANS hyperactivity, which over time will lead to increased pressure on the already weakened heart. Thus far, β-receptor blockade has been the main therapeutic approach with which the inhibition of the adrenergic drive has had a positive effect counteracting the progression of cardiovascular decline. With the emergence of new discoveries in stem cell research and their possible contribution to cardiac cell turnover and tissue repair, it is critical to evaluate the extent of the impact of the ANS on cardiac regeneration via the regulation of stem cells.

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.

Cardiovascular gene therapy is the third most popular application for gene therapy, representing 8.4% of all gene therapy trials as reported in 2012 estimates. Gene therapy in cardiovascular disease is aiming to treat heart failure from ischemic and non-ischemic causes, peripheral artery disease, venous ulcer, pulmonary hypertension, atherosclerosis and monogenic diseases, such as Fabry disease.In this review, we will focus on elucidating current molecular targets for the treatment of ventricular dysfunction following myocardial infarction (MI). In particular, we will focus on the treatment of i) the clinical consequences of it, such as heart failure and residual myocardial ischemia and ii) etiological causes of MI (coronary vessels atherosclerosis, bypass venous graft disease, in-stent restenosis).We summarise the scheme of the review and the molecular targets either already at the gene therapy clinical trial phase or in the pipeline. These targets will be discussed below. Following this, we will focus on what we believe are the 4 prerequisites of success of any gene target therapy: safety, expression, specificity and efficacy (SESE).

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.

Rationale: Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein–coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function. Objective: A loss of cardiac GRK2 activity is known to arrest progression of heart failure (HF), at least in part by normalization of cardiac β-adrenergic receptor (βAR) signaling. Chronic HF studies have been performed with GRK2 knockout mice, as well as expression of the βARKct, a peptide inhibitor of GRK2 activity. This study was conducted to examine the role of GRK2 and its activity during acute myocardial ischemic injury using an I/R model. Methods and Results: We demonstrate, using cardiac-specific GRK2 and βARKct-expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with βARKct imparting cardioprotection. Post-I/R infarct size was greater in GRK2-overexpressing mice (45.0±2.8% versus 31.3±2.3% in controls) and significantly smaller in βARKct mice (16.8±1.3%, P 2 ARs abolished βARKct-mediated cardioprotection, suggesting that enhanced GRK2 activity on this GPCR is deleterious to cardiac myocyte survival. Conclusion: The novel effect of reducing acute ischemic myocardial injury via increased Akt activity and NO production adds significantly to the therapeutic potential of GRK2 inhibition with the βARKct not only in chronic HF but also potentially in acute ischemic injury conditions.

Double-membrane-spanning gap junction (GJ) channels cluster into two-dimensional arrays, termed plaques, to provide direct cell-to-cell communication. GJ plaques often contain circular, channel-free domains ( approximately 0.05-0.5 mum in diameter) identified30 y ago and termed nonjunctional membrane (NM) domains. We show, by expressing the GJ protein connexin43 (Cx43) tagged with green fluorescent protein, or the novel photoconvertible fluorescent protein Dendra2, that NM domains appear to be remnants generated by the internalization of small GJ channel clusters that bud over time from central plaque areas. Channel clusters internalized within seconds forming endocytic double-membrane GJ vesicles ( approximately 0.18-0.27 mum in diameter) that were degraded by lysosomal pathways. Surprisingly, NM domains were not repopulated by surrounding channels and instead remained mobile, fused with each other, and were expelled at plaque edges. Quantification of internalized, photoconverted Cx43-Dendra2 vesicles indicated a GJ half-life of 2.6 h that falls within the estimated half-life of 1-5 h reported for GJs. Together with previous publications that revealed continuous accrual of newly synthesized channels along plaque edges and simultaneous removal of channels from plaque centers, our data suggest how the known dynamic channel replenishment of functional GJ plaques can be achieved. Our observations may have implications for the process of endocytic vesicle budding in general.

Gap junction channels may be comprised of either connexin or pannexin proteins (innexins and pannexins). Membrane topologies of both families are similar, but sequence similarity is lacking. Recently, connexin-like sequences have been identified in mammalian and zebrafish genomes that have only four conserved cysteines in the extracellular domains (Cx23), a feature of the pannexins. Phylogenetic analyses of the non-canonical “C4” connexins reveal that these sequences are indeed connexins. Functional assays reveal that the Cx23 gap junctions are capable of sharing neurobiotin, and further, that Cx23 connexins form hemichannels in vitro.

Monitoring Editor: Asma Nusrat Beyond its well-documented role in vesicle endocytosis, clathrin has also been implicated in the internalization of large particles such as viruses, pathogenic bacteria, and even latex beads. We have discovered an additional clathrin-dependent endocytic process that results in the internalization of large, double-membrane vesicles at lateral membranes of cells that are coupled by gap junctions (GJs). GJ channels bridge apposing cell membranes to mediate the direct transfer of electrical currents and signaling molecules from cell to cell. Here we report that entire GJ plaques, clusters of GJ channels, can be internalized to form large, double-membrane vesicles previously termed annular gap junctions (AGJs). These internalized AGJ vesicles subdivide into smaller vesicles that are degraded by endo-/lysosomal pathways. Mechanistic analyses revealed that clathrin-dependent endocytosis machinery-components, including clathrin itself, the alternative clathrin-adaptor Dab2, dynamin, myosin-VI, and actin are involved in the internalization, inward movement, and degradation of these large, intercellular double-membrane vesicles. These findings contribute to the understanding of clathrin's numerous emerging functions.

Direct cell–cell communication mediated by plasma membrane-spanning gap junction (GJ) channels is vital to all aspects of cellular life. Obviously, GJ intercellular communication (GJIC) requires precise regulation, and it is known that controlled biosynthesis and degradation, and channel opening and closing (gating) are exploited. We discovered that cells internalize GJs in response to various stimuli. Here, we report that GJ internalization is a clathrin-mediated endocytic process that utilizes the vesicle-coat protein clathrin, the adaptor proteins adaptor protein complex 2 and disabled 2, and the GTPase dynamin. To our knowledge, we are first to report that the endocytic clathrin machinery can internalize double-membrane vesicles into cells.

During the inflammatory response, activation of G-protein coupled receptors (GPCRs) by inflammatory mediators rapidly leads to inhibition of gap junction intercellular communication (GJIC); however, the steps that lead to this inhibition are not known. Combining high-resolution fluorescence microscopy and functional assays, we found that activation of the GPCRs PAR-1 and ETA/B by their natural inflammatory mediator agonists, thrombin and endothelin-1, resulted in rapid and acute internalization of gap junctions (GJs) that coincided with the inhibition of GJIC followed by increased vascular permeability. The endocytosis protein clathrin and the scaffold protein ZO-1 appeared to be involved in GJ internalization, and ZO-1 was partially displaced from GJs during the internalization process. These findings demonstrate that GJ internalization is an efficient mechanism for modulating GJIC in inflammatory response.