miR-182/183-Rasa1 axis induced macrophage polarization and redox regulation promotes repair after ischemic cardiac injury.

Few therapies have produced significant improvement in cardiac structure and function after ischemic cardiac injury (ICI). Our possible explanation is activation of local inflammatory responses negatively impact the cardiac repair process following ischemic injury. Factors that can alter immune response, including significantly altered cytokine levels in plasma and polarization of macrophages and T cells towards a pro-reparative phenotype in the myocardium post-MI is a valid strategy for reducing infarct size and damage after myocardial injury. Our previous studies showed that cortical bone stem cells (CBSCs) possess reparative effects after ICI. In our current study, we have identified that the beneficial effects of CBSCs appear to be mediated by miRNA in their extracellular vesicles (CBSC-EV). Our studies showed that CBSC-EV treated animals demonstrated reduced scar size, attenuated structural remodeling, and improved cardiac function versus saline treated animals. These effects were linked to the alteration of immune response, with significantly altered cytokine levels in plasma, and polarization of macrophages and T cells towards a pro-reparative phenotype in the myocardium post-MI. Our detailed in vitro studies demonstrated that CBSC-EV are enriched in miR-182/183 that mediates the pro-reparative polarization and metabolic reprogramming in macrophages, including enhanced OXPHOS rate and reduced ROS, via Ras p21 protein activator 1 (RASA1) axis under Lipopolysaccharides (LPS) stimulation. In summary, CBSC-EV deliver unique molecular cargoes, such as enriched miR-182/183, that modulate the immune response after ICI by regulating macrophage polarization and metabolic reprogramming to enhance repair.

Effects of maternal hypothyroidism on postnatal cardiomyocyte proliferation and cardiac disease responses of the progeny.

Maternal hypothyroidism (MH) could adversely affect the cardiac disease responses of the progeny. This study tested the hypothesis that MH reduces early postnatal cardiomyocyte (CM) proliferation so that the adult heart of MH progeny has a smaller number of larger cardiac myocytes, which imparts adverse cardiac disease responses following injury. Thyroidectomy (TX) was used to establish MH. The progeny from mice that underwent sham or TX surgery were termed Ctrl (control) or MH (maternal hypothyroidism) progeny, respectively. MH progeny had similar heart weight (HW) to body weight (BW) ratios and larger CM size consistent with fewer CMs at postnatal day 60 (P60) compared with Ctrl (control) progeny. MH progeny had lower numbers of EdU+, Ki67+, and phosphorylated histone H3 (PH3)+ CMs, which suggests they had a decreased CM proliferation in the postnatal timeframe. RNA-seq data showed that genes related to DNA replication were downregulated in P5 MH hearts, including bone morphogenetic protein 10 (Bmp10). Both in vivo and in vitro studies showed Bmp10 treatment increased CM proliferation. After transverse aortic constriction (TAC), the MH progeny had more severe cardiac pathological remodeling compared with the Ctrl progeny. Thyroid hormone (T4) treatment for MH mothers preserved their progeny's postnatal CM proliferation capacity and prevented excessive pathological remodeling after TAC. Our results suggest that CM proliferation during early postnatal development was significantly reduced in MH progeny, resulting in fewer CMs with hypertrophy in adulthood. These changes were associated with more severe cardiac disease responses after pressure overload.NEW & NOTEWORTHY Our study shows that compared with Ctrl (control) progeny, the adult progeny of mothers who have MH (MH progeny) had fewer CMs. This reduction of CM numbers was associated with decreased postnatal CM proliferation. Gene expression studies showed a reduced expression of Bmp10 in MH progeny. Bmp10 has been linked to myocyte proliferation. In vivo and in vitro studies showed that Bmp10 treatment of MH progeny and their myocytes could increase CM proliferation. Differences in CM number and size in adult hearts of MH progeny were linked to more severe cardiac structural and functional remodeling after pressure overload. T4 (synthetic thyroxine) treatment of MH mothers during their pregnancy, prevented the reduction in CM number in their progeny and the adverse response to disease stress.

Systemic Hypoxemia Induces Cardiomyocyte Hypertrophy and Right Ventricular Specific Induction of Proliferation.

BACKGROUND: A recent study suggests that systemic hypoxemia in adult male mice can induce cardiac myocytes to proliferate. The goal of the present experiments was to confirm these results, provide new insights on the mechanisms that induce adult cardiomyocyte cell cycle reentry, and to determine if hypoxemia also induces cardiomyocyte proliferation in female mice. METHODS: EdU-containing mini pumps were implanted in 3-month-old, male and female C57BL/6 mice. Mice were placed in a hypoxia chamber, and the oxygen was lowered by 1% every day for 14 days to reach 7% oxygen. The animals remained in 7% oxygen for 2 weeks before terminal studies. Myocyte proliferation was also studied with a mosaic analysis with double markers mouse model. RESULTS: Hypoxia induced cardiac hypertrophy in both left ventricular (LV) and right ventricular (RV) myocytes, with LV myocytes lengthening and RV myocytes widening and lengthening. Hypoxia induced an increase (0.01±0.01% in normoxia to 0.11±0.09% in hypoxia) in the number of EdU+ RV cardiomyocytes, with no effect on LV myocytes in male C57BL/6 mice. Similar results were observed in female mice. Furthermore, in mosaic analysis with double markers mice, hypoxia induced a significant increase in RV myocyte proliferation (0.03±0.03% in normoxia to 0.32±0.15% in hypoxia of RFP+ myocytes), with no significant change in LV myocyte proliferation. RNA sequencing showed upregulation of mitotic cell cycle genes and a downregulation of Cullin genes, which promote the G1 to S phase transition in hypoxic mice. There was significant proliferation of nonmyocytes and mild cardiac fibrosis in hypoxic mice that did not disrupt cardiac function. Male and female mice exhibited similar gene expression following hypoxia. CONCLUSIONS: Systemic hypoxia induces a global hypertrophic stress response that was associated with increased RV proliferation, and while LV myocytes did not show increased proliferation, our results minimally confirm previous reports that hypoxia can induce cardiomyocyte cell cycle activity in vivo.

Combining three independent pathological stressors induces a heart failure with preserved ejection fraction phenotype.

Heart failure (HF) with preserved ejection fraction (HFpEF) is defined as HF with an ejection fraction (EF) ≥ 50% and elevated cardiac diastolic filling pressures. The underlying causes of HFpEF are multifactorial and not well-defined. A transgenic mouse with low levels of cardiomyocyte (CM)-specific inducible Cavß2a expression (ß2a-Tg mice) showed increased cytosolic CM Ca2+, and modest levels of CM hypertrophy, and fibrosis. This study aimed to determine if ß2a-Tg mice develop an HFpEF phenotype when challenged with two additional stressors, high-fat diet (HFD) and Nω-nitro-l-arginine methyl ester (l-NAME, LN). Four-month-old wild-type (WT) and ß2a-Tg mice were given either normal chow (WT-N, ß2a-N) or HFD and/or l-NAME (WT-HFD, WT-LN, WT-HFD-LN, ß2a-HFD, ß2a-LN, and ß2a-HFD-LN). Some animals were treated with the histone deacetylase (HDAC) (hypertrophy regulators) inhibitor suberoylanilide hydroxamic acid (SAHA) (ß2a-HFD-LN-SAHA). Echocardiography was performed monthly. After 4 mo of treatment, terminal studies were performed including invasive hemodynamics and organs weight measurements. Cardiac tissue was collected. Four months of HFD plus l-NAME treatment did not induce a profound HFpEF phenotype in FVB WT mice. ß2a-HFD-LN (3-Hit) mice developed features of HFpEF, including increased atrial natriuretic peptide (ANP) levels, preserved EF, diastolic dysfunction, robust CM hypertrophy, increased M2-macrophage population, and myocardial fibrosis. SAHA reduced the HFpEF phenotype in the 3-Hit mouse model, by attenuating these effects. The 3-Hit mouse model induced a reliable HFpEF phenotype with CM hypertrophy, cardiac fibrosis, and increased M2-macrophage population. This model could be used for identifying and preclinical testing of novel therapeutic strategies.NEW & NOTEWORTHY Our study shows that three independent pathological stressors (increased Ca2+ influx, high-fat diet, and l-NAME) together produce a profound HFpEF phenotype. The primary mechanisms include HDAC-dependent-CM hypertrophy, necrosis, increased M2-macrophage population, fibroblast activation, and myocardial fibrosis. A role for HDAC activation in the HFpEF phenotype was shown in studies with SAHA treatment, which prevented the severe HFpEF phenotype. This "3-Hit" mouse model could be helpful in identifying novel therapeutic strategies to treat HFpEF.

RNA-Binding Protein LIN28a Regulates New Myocyte Formation in the Heart Through Long Noncoding RNA-H19.

BACKGROUND: Developmental cardiac tissue holds remarkable capacity to regenerate after injury and consists of regenerative mononuclear diploid cardiomyocytes. On maturation, mononuclear diploid cardiomyocytes become binucleated or polyploid and exit the cell cycle. Cardiomyocyte metabolism undergoes a profound shift that coincides with cessation of regeneration in the postnatal heart. However, whether reprogramming metabolism promotes persistence of regenerative mononuclear diploid cardiomyocytes enhancing cardiac function and repair after injury is unknown. Here, we identify a novel role for RNA-binding protein LIN28a, a master regulator of cellular metabolism in cardiac repair after injury. METHODS: LIN28a overexpression was tested using mouse transgenesis on postnatal cardiomyocyte numbers, cell cycle, and response to apical resection injury. With the use of neonatal and adult cell culture systems and adult and Mosaic Analysis with Double Markers myocardial injury models in mice, the effect of LIN28a overexpression on cardiomyocyte cell cycle and metabolism was tested. Last, isolated adult cardiomyocytes from LIN28a and wild-type mice 4 days after myocardial injury were used for RNA-immunoprecipitation sequencing. RESULTS: LIN28a was found to be active primarily during cardiac development and rapidly decreases after birth. LIN28a reintroduction at postnatal day (P) 1, P3, P5, and P7 decreased maturation-associated polyploidization, nucleation, and cell size, enhancing cardiomyocyte cell cycle activity in LIN28a transgenic pups compared with wild-type littermates. Moreover, LIN28a overexpression extended cardiomyocyte cell cycle activity beyond P7 concurrent with increased cardiac function 30 days after apical resection. In the adult heart, LIN28a overexpression attenuated cardiomyocyte apoptosis, enhanced cell cycle activity, cardiac function, and survival in mice 12 weeks after myocardial infarction compared with wild-type littermate controls. Instead, LIN28a small molecule inhibitor attenuated the proreparative effects of LIN28a on the heart. Neonatal rat ventricular myocytes overexpressing LIN28a mechanistically showed increased glycolysis, ATP production, and levels of metabolic enzymes compared with control. LIN28a immunoprecipitation followed by RNA-immunoprecipitation sequencing in cardiomyocytes isolated from LIN28a-overexpressing hearts after injury identified long noncoding RNA-H19 as its most significantly altered target. Ablation of long noncoding RNA-H19 blunted LIN28a-induced enhancement on cardiomyocyte metabolism and cell cycle activity. CONCLUSIONS: Collectively, LIN28a reprograms cardiomyocyte metabolism and promotes persistence of mononuclear diploid cardiomyocytes in the injured heart, enhancing proreparative processes, thereby linking cardiomyocyte metabolism to regulation of ploidy/nucleation and repair in the heart.

Sex-specific responses to slow progressive pressure overload in a large animal model of HFpEF.

Approximately 50% of all heart failure (HF) diagnoses can be classified as HF with preserved ejection fraction (HFpEF). HFpEF is more prevalent in females compared with males, but the underlying mechanisms are unknown. We previously showed that pressure overload (PO) in male felines induces a cardiopulmonary phenotype with essential features of human HFpEF. The goal of this study was to determine if slow progressive PO induces distinct cardiopulmonary phenotypes in females and males in the absence of other pathological stressors. Female and male felines underwent aortic constriction (banding) or sham surgery after baseline echocardiography, pulmonary function testing, and blood sampling. These assessments were repeated at 2 and 4 mo postsurgery to document the effects of slow progressive pressure overload. At 4 mo, invasive hemodynamic studies were also performed. Left ventricle (LV) tissue was collected for histology, myofibril mechanics, extracellular matrix (ECM) mass spectrometry, and single-nucleus RNA sequencing (snRNAseq). The induced pressure overload (PO) was not different between sexes. PO also induced comparable changes in LV wall thickness and myocyte cross-sectional area in both sexes. Both sexes had preserved ejection fraction, but males had a slightly more robust phenotype in hemodynamic and pulmonary parameters. There was no difference in LV fibrosis and ECM composition between banded male and female animals. LV snRNAseq revealed changes in gene programs of individual cell types unique to males and females after PO. Based on these results, both sexes develop cardiopulmonary dysfunction but the phenotype is somewhat less advanced in females.NEW & NOTEWORTHY We performed a comprehensive assessment to evaluate the effects of slow progressive pressure overload on cardiopulmonary function in a large animal model of heart failure with preserved ejection fraction (HFpEF) in males and females. Functional and structural assessments were performed at the organ, tissue, cellular, protein, and transcriptional levels. This is the first study to compare snRNAseq and ECM mass spectrometry of HFpEF myocardium from males and females. The results broaden our understanding of the pathophysiological response of both sexes to pressure overload. Both sexes developed a robust cardiopulmonary phenotype, but the phenotype was equal or a bit less robust in females.

UCP2 modulates cardiomyocyte cell cycle activity, acetyl-CoA, and histone acetylation in response to moderate hypoxia.

Developmental cardiac tissue is regenerative while operating under low oxygen. After birth, ambient oxygen is associated with cardiomyocyte cell cycle exit and regeneration. Likewise, cardiac metabolism undergoes a shift with cardiac maturation. Whether there are common regulators of cardiomyocyte cell cycle linking metabolism to oxygen tension remains unknown. The objective of the study is to determine whether mitochondrial UCP2 is a metabolic oxygen sensor regulating cardiomyocyte cell cycle. Neonatal rat ventricular myocytes (NRVMs) under moderate hypoxia showed increased cell cycle activity and UCP2 expression. NRVMs exhibited a metabolic shift toward glycolysis, reducing citrate synthase, mtDNA, mitochondrial membrane potential (ΔΨm), and DNA damage/oxidative stress, while loss of UCP2 reversed this phenotype. Next, WT and mice from a global UCP2-KO mouse line (UCP2KO) kept under hypoxia for 4 weeks showed significant decline in cardiac function that was more pronounced in UCP2KO animals. Cardiomyocyte cell cycle activity was reduced, while fibrosis and DNA damage was significantly increased in UCP2KO animals compared with WT under hypoxia. Mechanistically, UCP2 increased acetyl-CoA levels and histone acetylation, and it altered chromatin modifiers linking metabolism to cardiomyocyte cell cycle under hypoxia. Here, we show a potentially novel role for mitochondrial UCP2 as an oxygen sensor regulating cardiomyocyte cell cycle activity, acetyl-CoA levels, and histone acetylation in response to moderate hypoxia.

Cortical bone stem cell-derived exosomes' therapeutic effect on myocardial ischemia-reperfusion and cardiac remodeling.

Heart failure is the one of the leading causes of death in the United States. Heart failure is a complex syndrome caused by numerous diseases, including severe myocardial infarction (MI). MI occurs after an occlusion of a cardiac artery causing downstream ischemia. MI is followed by cardiac remodeling involving extensive remodeling and fibrosis, which, if the original insult is severe or prolonged, can ultimately progress into heart failure. There is no "cure" for heart failure because therapies to regenerate dead tissue are not yet available. Previous studies have shown that in both post-MI and post-ischemia-reperfusion (I/R) models of heart failure, administration of cortical bone stem cell (CBSC) treatment leads to a reduction in scar size and improved cardiac function. Our first study investigated the ability of mouse CBSC-derived exosomes (mCBSC-dEXO) to recapitulate mouse CBSCs (mCBSC) therapeutic effects in a 24-h post-I/R model. This study showed that injection of mCBSCs and mCBSC-dEXOs into the ischemic region of an infarct had a protective effect against I/R injury. mCBSC-dEXOs recapitulated the effects of CBSC treatment post-I/R, indicating exosomes are partly responsible for CBSC's beneficial effects. To examine if exosomes decrease fibrotic activation, adult rat ventricular fibroblasts (ARVFs) and adult human cardiac fibroblasts (NHCFs) were treated with transforming growth factor ß (TGFß) to activate fibrotic signaling before treatment with mCBSC- and human CBSC (hCBSC)-dEXOs. hCBSC-dEXOs caused a 100-fold decrease in human fibroblast activation. To further understand the signaling mechanisms regulating the protective decrease in fibrosis, we performed RNA sequencing on the NHCFs after hCBSC-dEXO treatment. The group treated with both TGFß and exosomes showed a decrease in small nucleolar RNA (snoRNA), known to be involved with ribosome stability.NEW & NOTEWORTHY Our work is noteworthy due to the identification of factors within stem cell-derived exosomes (dEXOs) that alter fibroblast activation through the hereto-unknown mechanism of decreasing small nucleolar RNA (snoRNA) signaling within cardiac fibroblasts. The study also shows that the injection of stem cells or a stem-cell-derived exosome therapy at the onset of reperfusion elicits cardioprotection, emphasizing the importance of early treatment in the post-ischemia-reperfusion (I/R) wounded heart.

Cardiomyocyte Proliferation as a Source of New Myocyte Development in the Adult Heart.

Cardiac diseases such as myocardial infarction (MI) can lead to adverse remodeling and impaired contractility of the heart due to widespread cardiomyocyte death in the damaged area. Current therapies focus on improving heart contractility and minimizing fibrosis with modest cardiac regeneration, but MI patients can still progress to heart failure (HF). There is a dire need for clinical therapies that can replace the lost myocardium, specifically by the induction of new myocyte formation from pre-existing cardiomyocytes. Many studies have shown terminally differentiated myocytes can re-enter the cell cycle and divide through manipulations of the cardiomyocyte cell cycle, signaling pathways, endogenous genes, and environmental factors. However, these approaches result in minimal myocyte renewal or cardiomegaly due to hyperactivation of cardiomyocyte proliferation. Finding the optimal treatment that will replenish cardiomyocyte numbers without causing tumorigenesis is a major challenge in the field. Another controversy is the inability to clearly define cardiomyocyte division versus myocyte DNA synthesis due to limited methods. In this review, we discuss several studies that induced cardiomyocyte cell cycle re-entry after cardiac injury, highlight whether cardiomyocytes completed cytokinesis, and address both limitations and methodological advances made to identify new myocyte formation.

Cardiac Remodeling During Pregnancy With Metabolic Syndrome: Prologue of Pathological Remodeling.

BACKGROUND: The heart undergoes physiological hypertrophy during pregnancy in healthy individuals. Metabolic syndrome (MetS) is now prevalent in women of child-bearing age and might add risks of adverse cardiovascular events during pregnancy. The present study asks if cardiac remodeling during pregnancy in obese individuals with MetS is abnormal and whether this predisposes them to a higher risk for cardiovascular disorders. METHODS: The idea that MetS induces pathological cardiac remodeling during pregnancy was studied in a long-term (15 weeks) Western diet-feeding animal model that recapitulated features of human MetS. Pregnant female mice with Western diet (45% kcal fat)-induced MetS were compared with pregnant and nonpregnant females fed a control diet (10% kcal fat). RESULTS: Pregnant mice fed a Western diet had increased heart mass and exhibited key features of pathological hypertrophy, including fibrosis and upregulation of fetal genes associated with pathological hypertrophy. Hearts from pregnant animals with WD-induced MetS had a distinct gene expression profile that could underlie their pathological remodeling. Concurrently, pregnant female mice with MetS showed more severe cardiac hypertrophy and exacerbated cardiac dysfunction when challenged with angiotensin II/phenylephrine infusion after delivery. CONCLUSIONS: These results suggest that preexisting MetS could disrupt physiological hypertrophy during pregnancy to produce pathological cardiac remodeling that could predispose the heart to chronic disorders.

Interaction of the Joining Region in Junctophilin-2 With the L-Type Ca2+ Channel Is Pivotal for Cardiac Dyad Assembly and Intracellular Ca2+ Dynamics.

RATIONALE: Ca2+-induced Ca2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules) and RyR (ryanodine receptors) within the junctional sarcoplasmic reticulum. CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. JPH (junctophilin) 2 enables close association between T-tubules and the junctional sarcoplasmic reticulum to ensure efficient CICR. JPH2 has a so-called joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. OBJECTIVE: To determine if the joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. METHODS AND RESULTS: Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult feline and rat ventricular myocytes. Protein-protein interaction studies showed that the joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mutPG1JPH2 caused asynchronous Ca2+-release with impaired excitation-contraction coupling after ß-adrenergic stimulation. The disturbed Ca2+ regulation in mutPG1JPH2 overexpressing myocytes caused calcium/calmodulin-dependent kinase II activation and altered myocyte bioenergetics. CONCLUSIONS: The interaction between LTCC and the joining region in JPH2 facilitates dyad assembly and maintains normal CICR in cardiomyocytes.

GDF11 Decreases Pressure Overload-Induced Hypertrophy, but Can Cause Severe Cachexia and Premature Death.

RATIONALE: Possible beneficial effects of GDF11 (growth differentiation factor 11) on the normal, diseased, and aging heart have been reported, including reversing aging-induced hypertrophy. These effects have not been well validated. High levels of GDF11 have also been shown to cause cardiac and skeletal muscle wasting. These controversies could be resolved if dose-dependent effects of GDF11 were defined in normal and aged animals as well as in pressure overload-induced pathological hypertrophy. OBJECTIVE: To determine dose-dependent effects of GDF11 on normal hearts and those with pressure overload-induced cardiac hypertrophy. METHODS AND RESULTS: Twelve- to 13-week-old C57BL/6 mice underwent transverse aortic constriction (TAC) surgery. One-week post-TAC, these mice received rGDF11 (recombinant GDF11) at 1 of 3 doses: 0.5, 1.0, or 5.0 mg/kg for up to 14 days. Treatment with GDF11 increased plasma concentrations of GDF11 and p-SMAD2 in the heart. There were no significant differences in the peak pressure gradients across the aortic constriction between treatment groups at 1 week post-TAC. Two weeks of GDF11 treatment caused dose-dependent decreases in cardiac hypertrophy as measured by heart weight/tibia length ratio, myocyte cross-sectional area, and left ventricular mass. GDF11 improved cardiac pump function while preventing TAC-induced ventricular dilation and caused a dose-dependent decrease in interstitial fibrosis (in vivo), despite increasing markers of fibroblast activation and myofibroblast transdifferentiation (in vitro). Treatment with the highest dose (5.0 mg/kg) of GDF11 caused severe body weight loss, with significant decreases in both muscle and organ weights and death in both sham and TAC mice. CONCLUSIONS: Although GDF11 treatment can reduce pathological cardiac hypertrophy and associated fibrosis while improving cardiac pump function in pressure overload, high doses of GDF11 cause severe cachexia and death. Use of GDF11 as a therapy could have potentially devastating actions on the heart and other tissues.

G protein-coupled receptor kinase 2 contributes to impaired fatty acid metabolism in the failing heart.

Increased G protein-coupled receptor kinase (GRK)2 is central to heart failure (HF) pathogenesis, via desensitization of ß-adrenergic receptors and loss of contractile reserve. Since GRK2 has been shown to compromise fatty acid (FA) oxidation, this kinase may link metabolic and contractile defects in HF. The aim of this study was to investigate the mechanistic role of GRK2 in FA metabolism and bioenergetics in the heart. For that purpose, we measured FA uptake and cluster of differentiation (CD)36 expression, phosphorylation, and ubiquitination in mice with cardiac-specific overexpression of GRK2 (TgGRK2) or expression of its c-terminus (GRK2 inhibitor- TgßARKct) or in global heterozygous GRK2 knockout (GRK2+/-) mice. Cellular bioenergetics were also measured in isolated cardiomyocytes following adenoviral delivery of exogenous GRK2, ßARKct, or short hairpin GRK2 (shGRK2). Additionally, CD36 expression and phosphorylation were evaluated following transverse aortic constriction (TAC) in wild type (WT) and GRK2+/- mice. Our results show a 33% ±â€¯0.81 reduction in FA uptake rate, accompanied by 51% ±â€¯0.17 lower CD36 protein, and 70% ±â€¯0.23 and 69% ±â€¯0.18 increases in CD36 phosphorylation and ubiquitination, respectively, in the TgGRK2 mice. Moreover, an in vitro kinase assay suggests that GRK2 directly phosphorylates CD36. In isolated cardiomyocytes, GRK2 overexpression induced a 26% ±â€¯2.21 decrease in maximal respiration, which was enhanced (20% ±â€¯4.02-5.14) with inhibition of the kinase. Importantly, in hearts with systolic dysfunction, notable reductions in CD36 mRNA and protein, as well as a significant increase in CD36 phosphorylation were normalized in the GRK2+/- mice post-TAC. Thus, we propose that GRK2 up-regulation in HF is, at least partly, responsible for reduced FA uptake and oxidation and may be a nodal link between metabolic and contractile defects.

Cortical Bone Stem Cell Therapy Preserves Cardiac Structure and Function After Myocardial Infarction.

RATIONALE: Cortical bone stem cells (CBSCs) have been shown to reduce ventricular remodeling and improve cardiac function in a murine myocardial infarction (MI) model. These effects were superior to other stem cell types that have been used in recent early-stage clinical trials. However, CBSC efficacy has not been tested in a preclinical large animal model using approaches that could be applied to patients. OBJECTIVE: To determine whether post-MI transendocardial injection of allogeneic CBSCs reduces pathological structural and functional remodeling and prevents the development of heart failure in a swine MI model. METHODS AND RESULTS: Female Göttingen swine underwent left anterior descending coronary artery occlusion, followed by reperfusion (ischemia-reperfusion MI). Animals received, in a randomized, blinded manner, 1:1 ratio, CBSCs (n=9; 2×107 cells total) or placebo (vehicle; n=9) through NOGA-guided transendocardial injections. 5-ethynyl-2'deoxyuridine (EdU)-a thymidine analog-containing minipumps were inserted at the time of MI induction. At 72 hours (n=8), initial injury and cell retention were assessed. At 3 months post-MI, cardiac structure and function were evaluated by serial echocardiography and terminal invasive hemodynamics. CBSCs were present in the MI border zone and proliferating at 72 hours post-MI but had no effect on initial cardiac injury or structure. At 3 months, CBSC-treated hearts had significantly reduced scar size, smaller myocytes, and increased myocyte nuclear density. Noninvasive echocardiographic measurements showed that left ventricular volumes and ejection fraction were significantly more preserved in CBSC-treated hearts, and invasive hemodynamic measurements documented improved cardiac structure and functional reserve. The number of EdU+ cardiac myocytes was increased in CBSC- versus vehicle- treated animals. CONCLUSIONS: CBSC administration into the MI border zone reduces pathological cardiac structural and functional remodeling and improves left ventricular functional reserve. These effects reduce those processes that can lead to heart failure with reduced ejection fraction.

Human polyoma JC virus minor capsid proteins, VP2 and VP3, enhance large T antigen binding to the origin of viral DNA replication: evidence for their involvement in regulation of the viral DNA replication.

JC virus (JCV) lytically infects the oligodendrocytes in the central nervous system in a subset of immunocompromized patients and causes the demyelinating disease, progressive multifocal leukoencephalopathy. JCV replicates and assembles into infectious virions in the nucleus. However, understanding the molecular mechanisms of its virion biogenesis remains elusive. In this report, we have attempted to shed more light on this process by investigating molecular interactions between large T antigen (LT-Ag), Hsp70 and minor capsid proteins, VP2/VP3. We demonstrated that Hsp70 interacts with VP2/VP3 and LT-Ag; and accumulates heavily in the nucleus of the infected cells. We also showed that VP2/VP3 associates with LT-Ag through their DNA binding domains resulting in enhancement in LT-Ag DNA binding to Ori and induction in viral DNA replication. Altogether, our results suggest that VP2/VP3 and Hsp70 actively participate in JCV DNA replication and may play critical roles in coupling of viral DNA replication to virion encapsidation.