PU.1 inhibition does not attenuate cardiac function deterioration or fibrosis in a murine model of myocardial infarction.

Activated cardiac fibroblasts are involved in both reparative wound healing and maladaptive cardiac fibrosis after myocardial infarction (MI). Recent evidence suggests that PU.1 inhibition can enable reprogramming of profibrotic fibroblasts to quiescent fibroblasts, leading to attenuation of pathologic fibrosis in several fibrosis models. The role of PU.1 in acute MI has not been tested. We designed a randomized, blinded study to evaluate whether DB1976, a PU.1 inhibitor, attenuates cardiac function deterioration and fibrosis in a murine model of MI. A total of 44 Ai9 periostin-Cre transgenic mice were subjected to 60 min of coronary occlusion followed by reperfusion. At 7 days after MI, 37 mice were randomly assigned to control (vehicle) or DB1976 treatment and followed for 2 weeks. Left ventricular ejection fraction (EF), assessed by echocardiography, did not differ between the two groups before or after treatment (final EF, 33.3 ± 1.0% in control group and 31.2 ± 1.3% in DB1976 group). Subgroup analysis of female and male mice showed the same results. There were no differences in cardiac scar (trichrome stain) and fibrosis (interstitial/perivascular collagen; picrosirius stain) between groups. Results from the per-protocol dataset (including mice with pre-treatment EF < 35% only) were consistent with the full dataset. In conclusion, this randomized, blinded study demonstrates that DB1976, a PU.1 inhibitor, does not attenuate cardiac functional deterioration or cardiac fibrosis in a mouse model of MI caused by coronary occlusion/reperfusion.

Effect of intravenous cell therapy in rats with old myocardial infarction.

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 12 × 106 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 days 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.

Single dose of synthetic microRNA-199a or microRNA-149 mimic does not improve cardiac function in a murine model of myocardial infarction.

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.

Comparison of Repeated Doses of C-kit-Positive Cardiac Cells versus a Single Equivalent Combined Dose in a Murine Model of Chronic Ischemic Cardiomyopathy.

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 < 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 < 0.05, in the risk region and -28.5%, 148.5 ± 12.1 µm2 vs. 207.6 ± 20.5 µm2, p < 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.

Cardiac mesenchymal cells from failing and nonfailing hearts limit ventricular dilation when administered late after infarction.

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.

E2f1 deletion attenuates infarct-induced ventricular remodeling without affecting O-GlcNAcylation.

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.

Epigenetically modified cardiac mesenchymal stromal cells limit myocardial fibrosis and promote functional recovery in a model of chronic ischemic cardiomyopathy.

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 naïve 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.

Leukocyte-Expressed ß2-Adrenergic Receptors Are Essential for Survival After Acute Myocardial Injury.

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 ß-adrenergic receptors (ßARs); 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 ßAR knockout (ßARKO) 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: ß2ARKO BMT mice displayed 100% mortality resulting from cardiac rupture within 12 days after MI compared with ≈20% mortality in WT BMT mice. ß2ARKO 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 ß-arrestin-dependent ß2AR signaling. Furthermore, vascular cell adhesion molecule-1 expression in both mouse and human macrophages was sensitive to ß2AR activity, and spleens from human tissue donors treated with ß-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 ß2AR in ß2ARKO bone marrow before transplantation, which also resulted in post-MI survival rates comparable to those in WT BMT mice. CONCLUSIONS: Immune cell-expressed ß2AR plays an essential role in regulating the early inflammatory repair response to acute myocardial injury by facilitating cardiac leukocyte infiltration.

Repeated doses of cardiac mesenchymal cells are therapeutically superior to a single dose in mice with old myocardial infarction.

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 < 0.05). The multiple-dose group also exhibited less collagen in the non-infarcted region vs. the single-dose group. Engraftment and differentiation of CMCs were negligible in both groups, indicating paracrine effects. These results demonstrate that, in mice with ischemic cardiomyopathy, the beneficial effects of three doses of CMCs are significantly greater than those of one dose, supporting the concept that multiple treatments are necessary to properly evaluate the full therapeutic potential of cell therapy. Thus, the repeated-treatment paradigm is not limited to c-kit POS CPCs or to rats, but applies to other cell types and species. The generalizability of this concept dramatically augments its significance.

Cardiomyocyte Ogt limits ventricular dysfunction in mice following pressure overload without affecting hypertrophy.

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 naïve 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.

Interleukin-10 inhibits chronic angiotensin II-induced pathological autophagy.

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.

Bone Marrow and Wharton's Jelly Mesenchymal Stromal Cells are Ineffective for Myocardial Repair in an Immunodeficient Rat Model of Chronic Ischemic Cardiomyopathy.

BACKGROUND: Although cell therapy provides benefits for outcomes of heart failure, the most optimal cell type to be used clinically remains unknown. Most of the cell products used for therapy in humans require in vitro expansion to obtain a suitable number of cells for treatment; however, the clinical background of the donor and limited starting material may result in the impaired proliferative and reparative capacity of the cells expanded in vitro. Wharton's jelly mesenchymal cells (WJ MSCs) provide a multitude of advantages over adult tissue-derived cell products for therapy. These include large starting tissue material, superior proliferative capacity, and disease-free donors. Thus, WJ MSC if effective would be the most optimal cell source for clinical use. OBJECTIVES: This study evaluated the therapeutic efficacy of Wharton's jelly (WJ) and bone marrow (BM) mesenchymal stromal cells (MSCs) in chronic ischemic cardiomyopathy in rats. METHODS: Human WJ MSCs and BM MSCs were expanded in vitro, characterized, and evaluated for therapeutic efficacy in a immunodeficient rat model of ischemic cardiomyopathy. Cardiac function was evaluated with hemodynamics and echocardiography. The extent of cardiac fibrosis, hypertrophy, and inflammation was assessed with histological analysis. RESULTS: In vitro analysis revealed that WJ MSCs and BM MSCs are morphologically and immunophenotypically indistinguishable. Nevertheless, the functional analysis showed that WJ MSCs have a superior proliferative capacity, less senescent phenotype, and distinct transcriptomic profile compared to BM MSC. WJ MSCs and BM MSC injected in rat hearts chronically after MI produced a small, but not significant improvement in heart structure and function. Histological analysis showed no difference in the scar size, collagen content, cardiomyocyte cross-sectional area, and immune cell count. CONCLUSIONS: Human WJ and BM MSC have a small but not significant effect on cardiac structure and function when injected intramyocardially in immunodeficient rats chronically after MI.

G protein-coupled receptor kinase 2 activity impairs cardiac glucose uptake and promotes insulin resistance after myocardial ischemia.

BACKGROUND: Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure. The chronic adrenergic stimulation that accompanies heart failure is a signaling abnormality that leads to the upregulation of G protein-coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part owing to uncoupling of the ß-adrenergic receptor system. In this study, we explored the possibility that enhanced GRK2 expression and activity, as seen during heart failure, can negatively affect cardiac metabolism as part of its pathogenic profile. METHODS AND RESULTS: Positron emission tomography studies revealed in transgenic mice that cardiac-specific overexpression of GRK2 negatively affected cardiac metabolism by inhibiting glucose uptake and desensitization of insulin signaling, which increases after ischemic injury and precedes heart failure development. Mechanistically, GRK2 interacts with and directly phosphorylates insulin receptor substrate-1 in cardiomyocytes, causing insulin-dependent negative signaling feedback, including inhibition of membrane translocation of the glucose transporter GLUT4. This identifies insulin receptor substrate-1 as a novel nonreceptor target for GRK2 and represents a new pathological mechanism for this kinase in the failing heart. Importantly, inhibition of GRK2 activity prevents postischemic defects in myocardial insulin signaling and improves cardiac metabolism via normalized glucose uptake, which appears to participate in GRK2-targeted prevention of heart failure. CONCLUSIONS: Our data provide novel insights into how GRK2 is pathological in the injured heart. Moreover, it appears to be a critical mechanistic link within neurohormonal crosstalk governing cardiac contractile signaling/function through ß-adrenergic receptors and metabolism through the insulin receptor.

Level of G protein-coupled receptor kinase-2 determines myocardial ischemia/reperfusion injury via pro- and anti-apoptotic mechanisms.

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 ß-adrenergic receptor (ßAR) signaling. Chronic HF studies have been performed with GRK2 knockout mice, as well as expression of the ßARKct, 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 ßARKct-expressing transgenic mice, a deleterious effect of GRK2 on in vivo myocardial I/R injury with ßARKct 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 ßARKct mice (16.8±1.3%, P<0.05). Importantly, in vivo apoptosis was found to be consistent with these reciprocal effects on post-I/R myocardial injury when levels of GRK2 activity were altered. Moreover, these results were reflected by higher Akt activation and induction of NO production via ßARKct, and these antiapoptotic/survival effects could be recapitulated in vitro. Interestingly, selective antagonism of ß(2)ARs abolished ßARKct-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 ßARKct not only in chronic HF but also potentially in acute ischemic injury conditions.

Comparison of One and Three Intraventricular Injections of Cardiac Progenitor Cells in a Murine Model of Chronic Ischemic Cardiomyopathy.

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 1st infusion (CPCs), but not after the 2nd and 3rd (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 < 0.05). Hemodynamic studies showed that compared with vehicle, both preload-dependent and preload-independent functional parameters were significantly increased in the multiple-dose group but not in the single-dose group. Thus, two independent methods of functional assessment (echocardiography and hemodynamic studies) consistently demonstrated the superiority of three doses of CPCs vs. one dose. Compared with the single-dose group, the multiple-dose group exhibited less LV hypertrophy, as evidenced by a greater reduction in LV/body weight ratio and cardiomyocyte cross-sectional area. Furthermore, unlike the single dose, three CPC doses reduced myocardial inflammatory cells in the risk region. This is the first study of echo-guided intraventricular infusion of CPCs in mice with chronic ischemic cardiomyopathy. The results demonstrate that the beneficial effects of three CPC doses are greater than those of one dose, supporting the concept that multiple treatments are necessary to properly evaluate cell therapy. Our findings indicate that this concept applies not only to rat models but also to murine models. The generalizability of this strategy greatly enhances its importance and provides a rationale for large animal studies. Graphical abstract.

Internalization of large double-membrane intercellular vesicles by a clathrin-dependent endocytic process.

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.

Cardiomyocyte Oga haploinsufficiency increases O-GlcNAcylation but hastens ventricular dysfunction following myocardial infarction.

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.

Cx23, a connexin with only four extracellular-loop cysteines, forms functional gap junction channels and hemichannels.

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.

Acute internalization of gap junctions in vascular endothelial cells in response to inflammatory mediator-induced G-protein coupled receptor activation.

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 ET(A/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.