Guo-Chang Fan

MicroRNA-320 Is Involved in the Regulation of Cardiac Ischemia/Reperfusion Injury by Targeting Heat-Shock Protein 20

Background— Recent studies have identified critical roles for microRNAs (miRNAs) in a variety of cellular processes, including regulation of cardiomyocyte death. However, the signature of miRNA expression and possible roles of miRNA in the ischemic heart have been less well studied. Methods and Results— We performed miRNA arrays to detect the expression pattern of miRNAs in murine hearts subjected to ischemia/reperfusion (I/R) in vivo and ex vivo. Surprisingly, we found that only miR-320 expression was significantly decreased in the hearts on I/R in vivo and ex vivo. This was further confirmed by TaqMan real-time polymerase chain reaction. Gain-of-function and loss-of-function approaches were employed in cultured adult rat cardiomyocytes to investigate the functional roles of miR-320. Overexpression of miR-320 enhanced cardiomyocyte death and apoptosis, whereas knockdown was cytoprotective, on simulated I/R. Furthermore, transgenic mice with cardiac-specific overexpression of miR-320 revealed an increased extent of apoptosis and infarction size in the hearts on I/R in vivo and ex vivo relative to the wild-type controls. Conversely, in vivo treatment with antagomir-320 reduced infarction size relative to the administration of mutant antagomir-320 and saline controls. Using TargetScan software and proteomic analysis, we identified heat-shock protein 20 (Hsp20), a known cardioprotective protein, as an important candidate target for miR-320. This was validated experimentally by utilizing a luciferase/GFP reporter activity assay and examining the expression of Hsp20 on miR-320 overexpression and knockdown in cardiomyocytes. Conclusions— Our data demonstrate that miR-320 is involved in the regulation of I/R-induced cardiac injury and dysfunction via antithetical regulation of Hsp20. Thus, miR-320 may constitute a new therapeutic target for ischemic heart diseases.

Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical difference between mouse and human

In human disease and experimental animal models, depressed Ca(2+) handling in failing cardiomyocytes is widely attributed to impaired sarcoplasmic reticulum (SR) function. In mice, disruption of the PLN gene encoding phospholamban (PLN) or expression of dominant-negative PLN mutants enhances SR and cardiac function, but effects of PLN mutations in humans are unknown. Here, a T116G point mutation, substituting a termination codon for Leu-39 (L39stop), was identified in two families with hereditary heart failure. The heterozygous individuals exhibited hypertrophy without diminished contractile performance. Strikingly, both individuals homozygous for L39stop developed dilated cardiomyopathy and heart failure, requiring cardiac transplantation at ages 16 and 27. An over 50% reduction in PLN mRNA and no detectable PLN protein were noted in one explanted heart. The expression of recombinant PLN-L39stop in human embryonic kidney (HEK) 293 cells and adult rat cardiomyocytes showed no PLN inhibition of SR Ca(2+)-ATPase and the virtual absence of stable PLN expression; where PLN was expressed, it was misrouted to the cytosol or plasma membrane. These findings describe a naturally-occurring loss-of-function human PLN mutation (PLN null). In contrast to reported benefits of PLN ablation in mouse heart failure, humans lacking PLN develop lethal dilated cardiomyopathy.

Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infarcted myocardium

Bone marrow mesenchymal stem cells (MSCs) participate in myocardial repair following myocardial infarction. However, their in vivo reparative capability is limited due to lack of their survival in the infarcted myocardium. To overcome this limitation, we genetically engineered male rat MSCs overexpressing CXCR4 in order to maximize the effect of stromal cell-derived factor-1alpha (SDF-1alpha) for cell migration and regeneration. MSCs were isolated from adult male rats and cultured. Adenoviral transduction was carried out to over-express either CXCR4/green fluorescent protein (Ad-CXCR4/GFP) or Ad-null/GFP alone (control). Flow cytometry was used to identify and isolate GFP/CXCR4 over-expressing MSCs for transplantation. Female rats were assigned to one of four groups (n=8 each) to receive GFP-transduced male MSCs (2 x 10(6)) via tail vein injection 3 days after ligation of the left anterior descending (LAD) coronary artery: GFP-transduced MSCs (Ad-null/GFP-MSCs, group 1) or MSCs over-expressing CXCR4/GFP (Ad-CXCR4/GFP-MSCs, group 2), or Ad-CXCR4/GFP-MSCs plus SDF-1alpha (50 ng/microl) (Ad-CXCR4/GFP-MSCs/SDF-1alpha, group 3), or Ad-miRNA targeting CXCR4 plus SDF-1alpha (Ad-miRNA/GFP-MSCs+SDF-1alpha treatment, group 4). Cardiodynamic data were obtained 4 weeks after induction of regional myocardial infarction (MI) using echocardiography after which hearts were harvested for immunohistochemical studies. The migration of GFP and Y-chromosome positive cells increased significantly in the peri- and infarct areas of groups 2 and 3 compared to control group (p<0.05), or miRNA-CXCR4 group (p<0.01). The number of CXCR4 positive cells in groups 2, 3 was intimately associated with angiogenesis and myogenesis. MSCs engraftment was blocked by pretreatment with miRNA (group 4). Cardiac function was significantly improved in rats receiving MSCs over-expressing CXCR4 alone or with SDF-1alpha. The up-regulation of matrix metalloproteinases (MMPs) by CXCR4 overexpressing MSCs perhaps facilitated their engraftment in the collagenous tissue of the infarcted area. CXCR4 over-expression led to enhance in vivo mobilization and engraftment of MSCs into ischemic area where these cells promoted neomyoangiogenesis and alleviated early signs of left ventricular remodeling.

A mutation in the human phospholamban gene, deleting arginine 14, results in lethal, hereditary cardiomyopathy

The sarcoplasmic reticulum Ca2+-cycling proteins are key regulators of cardiac contractility, and alterations in sarcoplasmic reticulum Ca2+-cycling properties have been shown to be causal of familial cardiomyopathies. Through genetic screening of dilated cardiomyopathy patients, we identified a previously uncharacterized deletion of arginine 14 (PLN-R14Del) in the coding region of the phospholamban (PLN) gene in a large family with hereditary heart failure. No homozygous individuals were identified. By middle age, heterozygous individuals developed left ventricular dilation, contractile dysfunction, and episodic ventricular arrhythmias, with overt heart failure in some cases. Transgenic mice overexpressing the mutant PLN-R14Del recapitulated human cardiomyopathy exhibiting similar histopathologic abnormalities and premature death. Coexpression of the normal and mutant-PLN in HEK-293 cells resulted in sarcoplasmic reticulum Ca2+-ATPase superinhibition. The dominant effect of the PLN-R14Del mutation could not be fully removed, even upon phosphorylation by protein kinase A. Thus, by chronic suppression of sarcoplasmic reticulum Ca2+-ATPase activity, the nonreversible superinhibitory function of mutant PLN-R14Del may lead to inherited dilated cardiomyopathy and premature death in both humans and mice.

MicroRNA-494 Targeting Both Proapoptotic and Antiapoptotic Proteins Protects Against Ischemia/Reperfusion-Induced Cardiac Injury

Background— MicroRNAs (miRs) participate in many cardiac pathophysiological processes, including ischemia/reperfusion (I/R)-induced cardiac injury. Recently, we and others observed that miR-494 was downregulated in murine I/R-injured and human infarcted hearts. However, the functional consequence of miR-494 in response to I/R remains unknown. Methods and Results— We generated a mouse model with cardiac-specific overexpression of miR-494. Transgenic hearts and wild-type hearts from multiple lines were subjected to global no-flow I/R with the Langendorff system. Transgenic hearts exhibited improved recovery of contractile performance over the reperfusion period. This improvement was accompanied by remarkable decreases in both lactate dehydrogenase release and the extent of apoptosis in transgenic hearts compared with wild-type hearts. In addition, myocardial infarction size was significantly reduced in transgenic hearts on I/R in vivo compared with wild-type hearts. Similarly, short-term overexpression of miR-494 in cultured adult cardiomyocytes demonstrated an inhibition of caspase-3 activity and reduced cell death on simulated I/R. In vivo treatment with antisense oligonucleotide miR-494 increased I/R-triggered cardiac injury relative to the administration of mutant antisense oligonucleotide miR-494 and saline controls. We further identified that 3 proapoptotic proteins (PTEN, ROCK1, and CaMKII&dgr;) and 2 antiapoptotic proteins (FGFR2 and LIF) were authentic targets for miR-494. Importantly, the Akt-mitochondrial signaling pathway was activated in miR-494–overexpressing myocytes. Conclusions— Our findings suggest that although miR-494 targets both proapoptotic and antiapoptotic proteins, the ultimate consequence is activation of the Akt pathway, leading to cardioprotective effects against I/R-induced injury. Thus, miR-494 may constitute a new therapeutic agent for the treatment of ischemic heart disease.

Novel Cardioprotective Role of a Small Heat-Shock Protein, Hsp20, Against Ischemia/Reperfusion Injury

Background—Heat-shock proteins (Hsps) have been shown to render cardioprotection from stress-induced injury; however, little is known about the role of another small heat-shock protein, Hsp20, which regulates activities of vasodilation and platelet aggregation, in cardioprotection against ischemia injury. We recently reported that increased expression of Hsp20 in cardiomyocytes was associated with improved contraction and protection against &bgr;-agonist–induced apoptosis. Methods and Results—To investigate whether overexpression of Hsp20 exerts protective effects in both ex vivo and in vivo ischemia/reperfusion (I/R) injury, we generated a transgenic (TG) mouse model with cardiac-specific overexpression of Hsp20 (10-fold). TG and wild-type (WT) hearts were then subjected to global no-flow I/R (45 minutes/120 minutes) using the Langendorff preparation. TG hearts exhibited improved recovery of contractile performance over the whole reperfusion period. This improvement was accompanied by a 2-fold decrease in lactate dehydrogenase released from the TG hearts. The extent of infarction and apoptotic cell death was also significantly decreased, which was associated with increased protein ratio of Bcl-2/Bax and reduced caspase-3 activity in TG hearts. Furthermore, in vivo experiments of 30-minute myocardial ischemia, via coronary artery occlusion, followed by 24-hour reperfusion, showed that the infarct region–to–risk region ratio was 8.1±1.1% in TG hearts (n=7), compared with 19.5±2.1% in WT hearts (n=11, P<0.001). Conclusions—Our data demonstrate that increased Hsp20 expression in the heart protects against I/R injury, resulting in improved recovery of cardiac function and reduced infarction. Thus, Hsp20 may constitute a new therapeutic target for ischemic heart diseases.

Hsp20-Engineered Mesenchymal Stem Cells Are Resistant to Oxidative Stress via Enhanced Activation of Akt and Increased Secretion of Growth Factors

Although heat‐shock preconditioning has been shown to promote cell survival under oxidative stress, the nature of heat‐shock response from different cells is variable and complex. Therefore, it remains unclear whether mesenchymal stem cells (MSCs) modified with a single heat‐shock protein (Hsp) gene are effective in the repair of a damaged heart. In this study, we genetically engineered rat MSCs with Hsp20 gene (Hsp20‐MSCs) and examined cell survival, revascularization, and functional improvement in rat left anterior descending ligation (LAD) model via intracardial injection. We observed that overexpression of Hsp20 protected MSCs against cell death triggered by oxidative stress in vitro. The survival of Hsp20‐MSCs was increased by approximately twofold by day 4 after transplantation into the infarcted heart, compared with that of vector‐MSCs. Furthermore, Hsp20‐MSCs improved cardiac function of infarcted myocardium as compared with vector‐MSCs, accompanied by reduction of fibrosis and increase in the vascular density. The mechanisms contributing to the beneficial effects of Hsp20 were associated with enhanced Akt activation and increased secretion of growth factors (VEGF, FGF‐2, and IGF‐1). The paracrine action of Hsp20‐MSCs was further validated in vitro by cocultured adult rat cardiomyocytes with a stress‐conditioned medium from Hsp20‐MSCs. Taken together, these data support the premise that genetic modification of MSCs before transplantation could be salutary for treating myocardial infarction. STEM CELLS 2009;27:3021–3031

Peripheral Nociception Associated With Surgical Incision Elicits Remote Nonischemic Cardioprotection Via Neurogenic Activation of Protein Kinase C Signaling

Background— Although remote ischemic stimuli have been shown to elicit cardioprotection against ischemia/reperfusion injury, there is little known about the effects of nonischemic stimuli. We previously described a remote cardioprotective effect of nonischemic surgical trauma (abdominal incision) called remote preconditioning of trauma (RPCT). In the present study, we elucidate mechanisms underlying this phenomenon. Methods and Results— We used a murine model of myocardial infarction to evaluate ischemia/reperfusion injury, and either abdominal surgical incision, or application of topical capsaicin, to elicit cardioprotection. We show that the cardioprotective effect of RPCT is initiated by skin nociception, and requires neurogenic signaling involving spinal nerves and activation of cardiac sensory and sympathetic nerves. Our results demonstrate bradykinin-dependent activation and repression, respectively, of PKCϵ and PKC&dgr; in myocardium after RPCT, and we show involvement of the KATP channels in cardioprotection. Finally, we show that topical application of capsaicin, which selectively activates C sensory fibers in the skin, mimics the cardioprotective effect of RPCT against myocardial infarction. Conclusions— Nontraumatic nociceptive preconditioning represents a novel therapeutic strategy for cardioprotection with great potential clinical utility.

Cardiomyocytes mediate anti-angiogenesis in type 2 diabetic rats through the exosomal transfer of miR-320 into endothelial cells

Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play critical roles in mediating cell-to-cell communication. Given that diabetic hearts exhibit insufficient angiogenesis, it is significant to test whether diabetic cardiomyocyte-derived exosomes possess any capacity in regulating angiogenesis. In this study, we first observed that both proliferation and migration of mouse cardiac endothelial cells (MCECs) were inhibited when co-cultured with cardiomyocytes isolated from adult Goto-Kakizaki (GK) rats, a commonly used animal model of type 2 diabetes. However, GK-myocyte-mediated anti-angiogenic effects were negated upon addition of GW4869, an inhibitor of exosome formation/release, into the co-cultures. Next, exosomes were purified from the myocyte culture supernatants by differential centrifugation. While exosomes derived from GK myocytes (GK-exosomes) displayed similar size and molecular markers (CD63 and CD81) to those originated from the control Wistar rat myocytes (WT-exosomes), their regulatory role in angiogenesis is opposite. We observed that the MCEC proliferation, migration and tube-like formation were inhibited by GK-exosomes, but were promoted by WT-exosomes. Mechanistically, we found that GK-exosomes encapsulated higher levels of miR-320 and lower levels of miR-126 compared to WT-exosomes. Furthermore, GK-exosomes were effectively taken up by MCECs and delivered miR-320. In addition, transportation of miR-320 from myocytes to MCECs could be blocked by GW4869. Importantly, the exosomal miR-320 functionally down-regulated its target genes (IGF-1, Hsp20 and Ets2) in recipient MCECs, and overexpression of miR-320 inhibited MCEC migration and tube formation. GK exosome-mediated inhibitory effects on angiogenesis were removed by knockdown of miR-320. Together, these data indicate that cardiomyocytes exert an anti-angiogenic function in type 2 diabetic rats through exosomal transfer of miR-320 into endothelial cells. Thus, our study provides a novel mechanism underlying diabetes mellitus-induced myocardial vascular deficiency which may be caused by secretion of anti-angiogenic exosomes from cardiomyocyes.

Small Heat-Shock Protein Hsp20 Phosphorylation Inhibits β-Agonist-Induced Cardiac Apoptosis

Activation of the sympathetic nervous system is a common compensatory feature in heart failure, but sustained β-adrenergic activation induces cardiomyocyte death, leading to cardiac remodeling and dysfunction. In mouse cardiomyocytes, we recently reported that prolonged exposure to β-agonists is associated with transient increases in expression and phosphorylation of a small heat-shock protein, Hsp20. To determine the functional significance of Hsp20, we overexpressed this protein and its constitutively phosphorylated (S16D) or nonphosphorylated (S16A) mutant in adult rat cardiomyocytes. Hsp20 protected cardiomyocytes from apoptosis triggered by activation of the cAMP-PKA pathway, as indicated by decreases in the number of pyknotic nuclei, terminal deoxynucleotidyltransfer-ase-mediated dUTP nick-end labeling, and DNA laddering, which were associated with inhibition of caspase-3 activity. These protective effects were further increased by the constitutively phosphorylated Hsp20 mutant (S16D), which conferred full protection from apoptosis. In contrast, the nonphosphorylatable mutant (S16A) exhibited no antiapoptotic properties. Immunostaining studies and immunoprecipitations with Hsp20 or actin antibodies demonstrated that Hsp20 translocated to cytoskeleton and associated with actin on isoproterenol stimulation. These findings suggest that Hsp20 and its phosphorylation at Ser16 may provide cardioprotection against β-agonist–induced apoptosis. Thus, Hsp20 may represent a novel therapeutic target in the treatment of heart failure.