Cornel Badorff

Transdifferentiation of Blood-Derived Human Adult Endothelial Progenitor Cells Into Functionally Active Cardiomyocytes

Background—Further to promoting angiogenesis, cell therapy may be an approach for cardiac regeneration. Recent studies suggest that progenitor cells can transdifferentiate into other lineages. However, the transdifferentiation potential of endothelial progenitor cells (EPCs) is unknown. Methods and Results—EPCs were obtained from peripheral blood mononuclear cells of healthy adults or coronary artery disease (CAD) patients by cultivating with endothelial cell medium and growth factors. After 3 days, >95% of adherent cells were functionally and phenotypically EPCs. Diacetylated LDL–labeled EPCs were then cocultivated with rat cardiomyocytes for 6 days, resulting in significant increases of EPC cell length and size to a cardiomyocyte-like morphology. Biochemically, 9.94±1.39% and 5.04±1.09% of EPCs from healthy adults (n=15) or CAD patients (n=14, P <0.01 versus healthy adults), respectively, expressed &agr;-sarcomeric actinin as measured by flow cytometry. Immunocytochemistry showed that human EPCs expressed &agr;-sarcomeric actinin, cardiac troponin I (both with partial sarcomeric organization), atrial natriuretic peptide, and myocyte enhancer factor 2. Fluo 4 imaging demonstrated calcium transients synchronized with adjacent rat cardiomyocytes in transdifferentiated human EPCs. Single-cell microinjection of Lucifer yellow and calcein-AM labeling of cardiomyocytes demonstrated gap junctional communication between 51±7% of EPCs (16 hours after labeling, n=4) and cardiomyocytes. EPC transdifferentiation into cardiomyocytes was not observed with conditioned medium but in coculture with paraformaldehyde-fixed cardiomyocytes. Conclusions—EPCs from healthy volunteers and CAD patients can transdifferentiate in vitro into functionally active cardiomyocytes when cocultivated with rat cardiomyocytes. Cell-to-cell contact but not cellular fusion mediates EPC transdifferentiation. The therapeutic use of autologous EPCs may aid cardiomyocyte regeneration in patients with ischemic heart disease.

Assessment of the Tissue Distribution of Transplanted Human Endothelial Progenitor Cells by Radioactive Labeling

Background—Transplantation of endothelial progenitor cells (EPCs) improves vascularization and left ventricular function after experimental myocardial ischemia. However, tissue distribution of transplanted EPCs has not yet been monitored in living animals. Therefore, we tested whether radioactive labeling allows us to detect injected EPCs. Methods and Results—Human EPCs were isolated from peripheral blood, characterized by expression of endothelial marker proteins, and radioactively labeled with [111In]indium oxine. EPCs (106) were injected in athymic nude rats 24 hours after myocardial infarction (n=8) or sham operation (n=8). Scintigraphic images were acquired after 1, 24, 48, and 96 hours after EPC injection. Animals were then killed, and specific radioactivity was measured in different tissues. At 24 to 96 hours after intravenous injection of EPCs, ≈70% of the radioactivity was localized in the spleen and liver, with only ≈1% of the radioactivity identified in the heart of sham-operated animals. After myocardial infarction, the heart-to-muscle radioactivity ratio increased significantly, from 1.02±0.19 in sham-operated animals to 2.03±0.37 after intravenous administration of EPCs. Injection of EPCs into the left ventricular cavity increased this ratio profoundly, from 2.69±1.54 in sham-operated animals to 4.70±1.55 (P <0.05) in rats with myocardial infarction. Immunostaining of cryosections from infarcted hearts confirmed that EPCs homed predominantly to the infarct border zone. Conclusions—Although only a small proportion of radiolabeled EPCs are detected in nonischemic myocardium, myocardial infarction increases homing of transplanted EPCs in vivo profoundly. Radiolabeling might eventually provide an useful tool for monitoring the fate of transplanted progenitor cells and for clinical cell therapy.

Enteroviral protease 2A cleaves dystrophin: Evidence of cytoskeletal disruption in an acquired cardiomyopathy

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy, but the mechanism of this pathology is unknown. Mutations in cytoskeletal proteins such as dystrophin cause hereditary dilated cardiomyopathy, but it is unclear if similar mechanisms underlie acquired forms of heart failure. We demonstrate here that purified Coxsackievirus protease 2A cleaves dystrophin in vitro as predicted by computer analysis. Dystrophin is also cleaved during Coxsackievirus infection of cultured myocytes and in infected mouse hearts, leading to impaired dystrophin function. In vivo, dystrophin and the dystrophin-associated glycoproteins α-sarcoglycan and β-dystroglycan are morphologically disrupted in infected myocytes. We suggest a molecular mechanism through which enteroviral infection contributes to the pathogenesis of acquired forms of dilated cardiomyopathy.

Akt-dependent phosphorylation of p21(Cip1) regulates PCNA binding and proliferation of endothelial cells.

ABSTRACT The protein kinase Akt is activated by growth factors and promotes cell survival and cell cycle progression. Here, we demonstrate that Akt phosphorylates the cell cycle inhibitory protein p21Cip1 at Thr 145 in vitro and in intact cells as shown by in vitro kinase assays, site-directed mutagenesis, and phospho-peptide analysis. Akt-dependent phosphorylation of p21Cip1 at Thr 145 prevents the complex formation of p21Cip1 with PCNA, which inhibits DNA replication. In addition, phosphorylation of p21Cip1 at Thr 145 decreases the binding of the cyclin-dependent kinases Cdk2 and Cdk4 to p21Cip1 and attenuates the Cdk2 inhibitory activity of p21Cip1. Immunohistochemistry and biochemical fractionation reveal that the decrease of PCNA binding and regulation of Cdk activity by p21Cip1 phosphorylation is not caused by altered intracellular localization of p21Cip1. As a functional consequence, phospho-mimetic mutagenesis of Thr 145 reverses the cell cycle-inhibitory properties of p21Cip1, whereas the nonphosphorylatable p21Cip1 T145A construct arrests cells in G0 phase. These data suggest that the modulation of p21Cip1 cell cycle functions by Akt-mediated phosphorylation regulates endothelial cell proliferation in response to stimuli that activate Akt.

Glycogen synthase kinase-3 couples AKT-dependent signaling to the regulation of p21Cip1 degradation.

Signaling via the phosphoinositide 3-kinase (PI3K)/AKT pathway is crucial for the regulation of endothelial cell (EC) proliferation and survival, which involves the AKT-dependent phosphorylation of the DNA repair protein p21Cip1 at Thr-145. Because p21Cip1 is a short-lived protein with a high proteasomal degradation rate, we investigated the regulation of p21Cip1protein levels by PI3K/AKT-dependent signaling. The PI3K inhibitors Ly294002 and wortmannin reduced p21Cip1 protein abundance in human umbilical vein EC. However, mutation of the AKT site Thr-145 into aspartate (T145D) did not increase its protein half-life. We therefore investigated whether a kinase downstream of AKT regulates p21Cip1 protein levels. In various cell types, AKT phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). Upon serum stimulation of EC, GSK-3β was phosphorylated at Ser-9. Site-directed mutagenesis revealed that GSK-3 in vitrophosphorylated p21Cip1 specifically at Thr-57 within the Cdk binding domain. Overexpression of GSK-3β decreased p21Cip1 protein levels in EC, whereas the specific inhibition of GSK-3 with lithium chloride interfered with p21Cip1 degradation and increased p21Cip1protein about 10-fold in EC and cardiac myocytes (30 mm,p < 0.001). These data indicate that GSK-3 triggers p21Cip1 degradation. In contrast, stimulation of AKT increases p21Cip1 via inhibitory phosphorylation of GSK-3.

Fas receptor signaling inhibits glycogen synthase kinase 3β and induces cardiac hypertrophy following pressure overload

Congestive heart failure is a leading cause of mortality in developed countries. Myocardial hypertrophy resulting from hypertension often precedes heart failure. Understanding the signaling underlying cardiac hypertrophy and failure is of major interest. Here, we identified Fas receptor activation, a classical death signal causing apoptosis via activation of the caspase cascade in many cell types, as a novel pathway mediating cardiomyocyte hypertrophy in vitro and in vivo. Fas activation by Fas ligand induced a hypertrophic response in cultured cardiomyocytes, which was dependent on the inactivation of glycogen synthase kinase 3 beta (GSK3 beta) by phosphorylation. In vivo, lpr (lymphoproliferative disease) mice lacking a functional Fas receptor demonstrated rapid-onset left ventricular dilatation and failure, absence of compensatory hypertrophy, and significantly increased mortality in response to pressure overload induction that was accompanied by a failure to inhibit GSK3 beta activity. In contrast, Fas ligand was dispensable for the development of pressure overload hypertrophy in vivo. In vitro, neonatal cardiomyocytes from lpr mice showed a completely abrogated or significantly blunted hypertrophic response after stimulation with Fas ligand or angiotensin II, respectively. These findings indicate that Fas receptor signaling inhibits GSK3 beta activity in cardiomyocytes and is required for compensation of pressure overload in vivo.

Non-canonical Wnt Signaling Enhances Differentiation of Human Circulating Progenitor Cells to Cardiomyogenic Cells

Human endothelial circulating progenitor cells (CPCs) can differentiate to cardiomyogenic cells during co-culture with neonatal rat cardiomyocytes. Wnt proteins induce myogenic specification and cardiac myogenesis. Here, we elucidated the effect of Wnts on differentiation of CPCs to cardiomyogenic cells. CPCs from peripheral blood mononuclear cells were isolated from healthy volunteers and co-cultured with neonatal rat cardiomyocytes. 6–10 days after co-culture, cardiac differentiation was determined by α-sarcomeric actinin staining of human lymphocyte antigen-positive cells (fluorescence-activated cell-sorting analysis) and mRNA expression of human myosin heavy chain and atrial natriuretic peptide. Supplementation of co-cultures with Wnt11-conditioned medium significantly enhanced the differentiation of CPCs to cardiomyocytes (1.7 ± 0.3-fold), whereas Wnt3A-conditioned medium showed no effect. Cell fusion was not affected by Wnt11-conditioned medium. Because Wnts inhibit glycogen synthase kinase-3β, we further determined whether the glycogen synthase kinase-3β inhibitor LiCl also enhanced cardiac differentiation of CPCs. However, LiCl (10 mm) did not affect CPC differentiation. In contrast, Wnt11-conditioned medium time-dependently activated protein kinase C (PKC). Moreover, the PKC inhibitors bisindolylmaleimide I and III significantly blocked differentiation of CPCs to cardiomyocytes. PKC activation by phorbol 12-myristate 13-acetate significantly increased CPC differentiation to a similar extent as compared with Wnt11-conditioned medium. Our data demonstrate that Wnt11, but not Wnt3A, augments cardiomyogenic differentiation of human CPCs. Wnt11 promotes cardiac differentiation via the non-canonical PKC-dependent signaling pathway.

Dystrophin deficiency markedly increases enterovirus-induced cardiomyopathy: A genetic predisposition to viral heart disease

Both enteroviral infection of the heart and mutations in the dystrophin gene can cause cardiomyopathy. Little is known, however, about the interaction between genetic and acquired forms of cardiomyopathy. We previously demonstrated that the enteroviral protease 2A cleaves dystrophin; therefore, we hypothesized that dystrophin deficiency would predispose to enterovirus-induced cardiomyopathy. We observed more severe cardiomyopathy, worsening over time, and greater viral replication in dystrophin-deficient mice infected with enterovirus than in infected wild-type mice. This difference appears to be a result of more efficient release of the virus from dystrophin-deficient myocytes. In addition, we found that expression of wild-type dystrophin in cultured cells decreased the cytopathic effect of enteroviral infection and the release of virus from the cell. We also found that expression of a cleavage-resistant mutant dystrophin further inhibited the virally mediated cytopathic effect and viral release. These results indicate that viral infection can influence the severity and penetrance of the cardiomyopathy that occurs in the hearts of dystrophin-deficient individuals.

The Immune System in Viral Myocarditis: Maintaining the Balance

“Nature has provided in the white corpuscles as you call them—or the phagocytes as we call them—a natural means of devouring and destroying all disease germs. [However,] …the inoculation that ought to cure sometimes kills.” Bernard Shaw, The Doctor’s Dilemma , 1906 Dilated cardiomyopathy, one of the leading causes of heart failure in the United States, is a multifactorial disease that includes both hereditary and acquired forms.1 In patients, it has been shown that dilated cardiomyopathy can be a sequela of viral myocarditis.2 Although many different infectious agents have been attributed as the cause of viral myocarditis, enteroviruses, in particular Coxsackie B viruses, are consistently among the most common.2 The concept that enteroviruses contribute to the pathogenesis of a subset of human dilated cardiomyopathy has been strengthened by the detection of enteroviral genome in the hearts of patients with dilated cardiomyopathy. Since the first description by Bowles et al in 1986,3 many articles have addressed this issue (reviewed in Baboonian et al4 ). Although the results of individual studies vary, the data overall indicate that enteroviral genome is present in the heart of 15% to 25% of patients with dilated cardiomyopathy.4 Analogous to many other viral illnesses, both direct viral injury and the immune response of the host play an important role in the pathogenesis of viral heart disease. Furthermore, results from experiments in murine models of viral myocarditis indicate that although the immune response has an important protective role, it may also have deleterious effects on the host. The balance between these protective and deleterious effects may ultimately determine the course of disease after enteroviral infection. The direct viral effects have been demonstrated in culture and in vivo. In cultured cardiomyocytes, infection with Coxsackievirus B3 (CVB3) induces a direct cytopathic effect and cell …

Glycogen Synthase Kinase 3β Inhibits Myocardin-Dependent Transcription and Hypertrophy Induction Through Site-Specific Phosphorylation

Cardiomyocyte hypertrophy is transcriptionally controlled and inhibited by glycogen synthase kinase 3&bgr; (GSK3&bgr;). Myocardin is a muscle-specific transcription factor with yet unknown relation to hypertrophy. Therefore, we investigated whether myocardin is sufficient to induce cardiomyocyte hypertrophy and whether myocardin is regulated by GSK3&bgr; through site-specific phosphorylation. Adenoviral myocardin overexpression induced cardiomyocyte hypertrophy in neonatal rat cardiomyocytes, with increased cell size, total protein amount, and transcription of atrial natriuretic factor (ANF). In vitro and in vivo (HEK 293 cells) kinase assays with synthetic peptides and full-length myocardin demonstrated that myocardin was a “primed” GSK3&bgr; substrate, with serines 455 to 467 and 624 to 636 being the major GSK3&bgr; phosphorylation sites. Myocardin-induced ANF transcription and increase in total protein amount were enhanced by GSK3&bgr; blockade (10 mmol/L LiCl), indicating that GSK3&bgr; inhibits myocardin. A GSK3&bgr; phosphorylation-resistant myocardin mutant (8xA) activated ANF transcription twice as potently as wildtype myocardin under basal conditions with GSK3&bgr; being active. Conversely, a GSK3&bgr; phospho-mimetic myocardin mutant (8xD) was transcriptionally repressed after GSK3&bgr; blockade, indicating that GSK3&bgr; phosphorylation at the sites identified inhibits myocardin transcriptional activity. GAL4-myocardin fusion constructs demonstrated that GSK3&bgr; phosphorylation reduced the intrinsic myocardin transcriptional activity. A cell-permeable (Antennapedia protein transduction tag) peptide containing the mapped myocardin GSK3&bgr; motifs 624 to 636 induced hypertrophy of cultured cardiomyocytes, suggesting that the peptide acted as substrate-based GSK3&bgr; inhibitor in cardiomyocytes. Therefore, we conclude that the GSK3&bgr;–myocardin interaction constitutes a novel molecular control of cardiomyocyte hypertrophy. Phosphorylation by GSK3&bgr; comprises a novel post-transcriptional regulatory mechanism of myocardin.