Vinciane Gaussin

Cardiac progenitor cells from adult myocardium: Homing, differentiation, and fusion after infarction

Potential repair by cell grafting or mobilizing endogenous cells holds particular attraction in heart disease, where the meager capacity for cardiomyocyte proliferation likely contributes to the irreversibility of heart failure. Whether cardiac progenitors exist in adult myocardium itself is unanswered, as is the question whether undifferentiated cardiac precursor cells merely fuse with preexisting myocytes. Here we report the existence of adult heart-derived cardiac progenitor cells expressing stem cell antigen-1. Initially, the cells express neither cardiac structural genes nor Nkx2.5 but differentiate in vitro in response to 5′-azacytidine, in part depending on Bmpr1a, a receptor for bone morphogenetic proteins. Given intravenously after ischemia/reperfusion, cardiac stem cell antigen 1 cells home to injured myocardium. By using a Cre/Lox donor/recipient pair (αMHC-Cre/R26R), differentiation was shown to occur roughly equally, with and without fusion to host cells.

TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice.

The transforming-growth-factor-β-activated kinase TAK1 is a member of the mitogen-activated protein kinase kinase kinase family, which couples extracellular stimuli to gene transcription. The in vivo function of TAK1 is not understood. Here, we investigated the potential involvement of TAK1 in cardiac hypertrophy. In adult mouse myocardium, TAK1 kinase activity was upregulated 7 days after aortic banding, a mechanical load that induces hypertrophy and expression of transforming growth factor β. An activating mutation of TAK1 expressed in myocardium of transgenic mice was sufficient to produce p38 mitogen-activated protein kinase phosphorylation in vivo, cardiac hypertrophy, interstitial fibrosis, severe myocardial dysfunction, ‘fetal’ gene induction, apoptosis and early lethality. Thus, TAK1 activity is induced as a delayed response to mechanical stress, and can suffice to elicit myocardial hypertrophy and fulminant heart failure.

Endocardial cushion and myocardial defects after cardiac myocyte-specific conditional deletion of the bone morphogenetic protein receptor ALK3

Receptors for bone morphogenetic proteins (BMPs), members of the transforming growth factor-β (TGFβ) superfamily, are persistently expressed during cardiac development, yet mice lacking type II or type IA BMP receptors die at gastrulation and cannot be used to assess potential later roles in creation of the heart. Here, we used a Cre/lox system for cardiac myocyte-specific deletion of the type IA BMP receptor, ALK3. ALK3 was specifically required at mid-gestation for normal development of the trabeculae, compact myocardium, interventricular septum, and endocardial cushion. Cardiac muscle lacking ALK3 was specifically deficient in expressing TGFβ2, an established paracrine mediator of cushion morphogenesis. Hence, ALK3 is essential, beyond just the egg cylinder stage, for myocyte-dependent functions and signals in cardiac organogenesis.

Guided Cardiopoiesis Enhances Therapeutic Benefit of Bone Marrow Human Mesenchymal Stem Cells in Chronic Myocardial Infarction

OBJECTIVES The goal of this study was to guide bone marrow-derived human mesenchymal stem cells (hMSCs) into a cardiac progenitor phenotype and assess therapeutic benefit in chronic myocardial infarction. BACKGROUND Adult stem cells, delivered in their naïve state, demonstrate a limited benefit in patients with ischemic heart disease. Pre-emptive lineage pre-specification may optimize therapeutic outcome. METHODS hMSC were harvested from a coronary artery disease patient cohort. A recombinant cocktail consisting of transforming growth factor-beta(1), bone morphogenetic protein-4, activin A, retinoic acid, insulin-like growth factor-1, fibroblast growth factor-2, alpha-thrombin, and interleukin-6 was formulated to engage hMSC into cardiopoiesis. Derived hMSC were injected into the myocardium of a nude infarcted murine model and followed over 1 year for functional and structural end points. RESULTS Although the majority of patient-derived hMSC in their native state demonstrated limited effect on ejection fraction, stem cells from rare individuals harbored a spontaneous capacity to improve contractile performance. This reparative cytotype was characterized by high expression of homeobox transcription factor Nkx-2.5, T-box transcription factor TBX5, helix-loop-helix transcription factor MESP1, and myocyte enhancer factor MEF2C, markers of cardiopoiesis. Recombinant cardiogenic cocktail guidance secured the cardiopoietic phenotype across the patient cohort. Compared with unguided counterparts, cardiopoietic hMSC delivered into infarcted myocardium achieved superior functional and structural benefit without adverse side effects. Engraftment into murine hearts was associated with increased human-specific nuclear, sarcomeric, and gap junction content along with induction of myocardial cell cycle activity. CONCLUSIONS Guided cardiopoiesis thus enhances the therapeutic benefit of bone marrow-derived hMSC in chronic ischemic cardiomyopathy.

Program of Cell Survival Underlying Human and Experimental Hibernating Myocardium

Hibernating myocardium refers to chronically dysfunctional myocardium in patients with coronary artery disease in which cardiac viability is maintained and whose function improves after coronary revascularization. It is our hypothesis that long-term adaptive genomic mechanisms subtend the survival capacity of this ischemic myocardium. Therefore, the goal of this study was to determine whether chronic repetitive ischemia elicits a gene program of survival protecting hibernating myocardium against cell death. Accordingly, we measured the expression of survival genes in hibernating myocardium, both in patients surgically treated for hibernation and in a chronic swine model of repetitive ischemia reproducing the features of hibernation. Human hibernating myocardium was characterized by an upregulation of genes and corresponding proteins involved in anti-apoptosis (IAP), growth (VEGF, H11 kinase), and cytoprotection (HSP70, HIF-1&agr;, GLUT1). In the swine model, the same genes and proteins were upregulated after repetitive ischemia, which was accompanied by a concomitant decrease in myocyte apoptosis. These changes characterize viable tissue, because they were not found in irreversibly injured myocardium. Our report demonstrates a novel mechanism by which the activation of an endogenous gene program of cell survival underlies the sustained viability of the hibernating heart. Potentially, promoting such a program offers a novel opportunity to salvage postmitotic tissues in conditions of ischemia.

Alk3/Bmpr1a receptor is required for development of the atrioventricular canal into valves and annulus fibrosus

Endocardial cushions are precursors of mature atrioventricular (AV) valves. Their formation is induced by signaling molecules originating from the AV myocardium, including bone morphogenetic proteins (BMPs). Here, we hypothesized that BMP signaling plays an important role in the AV myocardium during the maturation of AV valves from the cushions. To test our hypothesis, we used a unique Cre/lox system to target the deletion of a floxed Alk3 allele, the type IA receptor for BMPs, to cardiac myocytes of the AV canal (AVC). Lineage analysis indicated that cardiac myocytes of the AVC contributed to the tricuspid mural and posterior leaflets, the mitral septal leaflet, and the atrial border of the annulus fibrosus. When Alk3 was deleted in these cells, defects were seen in the same leaflets, ie, the tricuspid mural leaflet and mitral septal leaflet were longer, the tricuspid posterior leaflet was displaced and adherent to the ventricular wall, and the annulus fibrosus was disrupted resulting in ventricular preexcitation. The defects seen in mice with AVC-targeted deletion of Alk3 provide strong support for a role of Alk3 in human congenital heart diseases, such as Ebstein’s anomaly. In conclusion, our mouse model demonstrated critical roles for Alk3 signaling in the AV myocardium during the development of AV valves and the annulus fibrosus.

Gene program for cardiac cell survival induced by transient ischemia in conscious pigs

Therapy for ischemic heart disease has been directed traditionally at limiting cell necrosis. We determined by genome profiling whether ischemic myocardium can trigger a genetic program promoting cardiac cell survival, which would be a novel and potentially equally important mechanism of salvage. Although cardiac genomics is usually performed in rodents, we used a swine model of ischemia/reperfusion followed by ventricular dysfunction (stunning), which more closely resembles clinical conditions. Gene expression profiles were compared by subtractive hybridization between ischemic and normal tissue of the same hearts. About one-third (23/74) of the nuclear-encoded genes that were up-regulated in ischemic myocardium participate in survival mechanisms (inhibition of apoptosis, cytoprotection, cell growth, and stimulation of translation). The specificity of this response was confirmed by Northern blot and quantitative PCR. Unexpectedly, this program also included genes not previously described in cardiomyocytes. Up-regulation of survival genes was more profound in subendocardium over subepicardium, reflecting that this response in stunned myocardium was proportional to the severity of the ischemic insult. Thus, in a swine model that recapitulates human heart disease, nonlethal ischemia activates a genomic program of cell survival that relates to the time course of myocardial stunning and differs transmurally in relation to ischemic stress, which induced the stunning. Understanding the genes up-regulated during myocardial stunning, including those not previously described in the heart, and developing strategies that activate this program may open new avenues for therapy in ischemic heart disease.

H11 kinase is a novel mediator of myocardial hypertrophy in vivo.

Abstract— By subtractive hybridization, we found a significant increase in H11 kinase transcript in large mammalian models of both ischemia/reperfusion (stunning) and chronic pressure overload with hypertrophy. Because this gene has not been characterized in the heart, the goal of the present study was to determine the function of H11 kinase in cardiac tissue, both in vitro and in vivo. In isolated neonatal rat cardiac myocytes, adenoviral-mediated overexpression of H11 kinase resulted in a 37% increase in protein/DNA ratio, reflecting hypertrophy. A cardiac-specific transgene driven by the &agr;MHC-promoter was generated, which resulted in an average 7-fold increase in H11 kinase protein expression. Transgenic hearts were characterized by a 30% increase of the heart weight/body weight ratio, by the reexpression of a fetal gene program, and by concentric hypertrophy with preserved contractile function at echocardiography. This phenotype was accompanied by a dose-dependent activation of Akt/PKB and p70S6 kinase, whereas the MAP kinase pathway was unaffected. Thus, H11 kinase represents a novel mediator of cardiac cell growth and hypertrophy.

Common Genomic Response in Different Mouse Models of β-Adrenergic–Induced Cardiomyopathy

Background—Although &bgr;-adrenergic receptor (AR) blockade therapy is beneficial in the treatment of heart failure, little is known regarding the transcriptional mechanisms underlying this salutary action. Methods and Results—In the present study, we screened mice overexpressing Gs&agr;, &bgr;1AR, &bgr;2AR, or protein kinase A to test if a common genomic pathway exists in different models with enhanced &bgr;-adrenergic signaling. In mice overexpressing Gs&agr;, differentially expressed genes were identified by mRNA profiling. In addition to well-known markers of cardiac hypertrophy (atrial natriuretic factor, CARP, and &bgr;-myosin heavy chain), uncoupling protein 2 (UCP2), a protein involved in the control of mitochondrial membrane potential, and four-and-a-half LIM domain protein-1 (FHL1), a member of the LIM protein family, were predicted to be upregulated. Upregulation of these genes was confirmed by quantitative reverse transcriptase–polymerase chain reaction at all time points tested during the development of cardiomyopathy in mice overexpressing Gs&agr;. In mice overexpressing &bgr;1AR, &bgr;2AR, or protein kinase A, increased UCP2 and FHL1 expression was also observed at the onset of cardiomyopathy. &bgr;AR blockade treatment reversed the cardiomyopathy and suppressed the increased expression of UCP2 and FHL1 in mice overexpressing Gs&agr;. Conclusions—UCP2 and FHL1 are important candidate genes that correlate with the development of &bgr;AR-induced cardiomyopathy in different mouse models with enhanced &bgr;AR signaling. In addition to preserving cardiac function, &bgr;AR blockade treatment also prevents the genomic regulation that correlates with the onset of heart failure.

H11 Kinase Prevents Myocardial Infarction by Preemptive Preconditioning of the Heart

Ischemic preconditioning confers powerful protection against myocardial infarction through pre-emptive activation of survival signaling pathways, but it remains difficult to apply to patients with ischemic heart disease, and its effects are transient. Promoting a sustained activation of preconditioning mechanisms in vivo would represent a novel approach of cardioprotection. We tested the role of the protein H11 kinase (H11K), which accumulates by 4- to 6-fold in myocardium of patients with chronic ischemic heart disease and in experimental models of ischemia. This increased expression was quantitatively reproduced in cardiac myocytes using a transgenic (TG) mouse model. After 45 minutes of coronary artery occlusion and reperfusion, hearts from TG mice showed an 82±5% reduction in infarct size compared with wild-type (WT), which was similar to the 84±4% reduction of infarct size observed in WT after a protocol of ischemic preconditioning. Hearts from TG mice showed significant activation of survival kinases participating in preconditioning, including Akt and the 5′AMP-activated protein kinase (AMPK). H11K directly binds to both Akt and AMPK and promotes their nuclear translocation and their association in a multiprotein complex, which results in a stimulation of survival mechanisms in cytosol and nucleus, including inhibition of proapoptotic effectors (glycogen synthase kinase-3β, Bad, and Foxo), activation of antiapoptotic effectors (protein kinase C&egr;, endothelial and inducible NO synthase isoforms, and heat shock protein 70), increased expression of the hypoxia-inducible factor-1α, and genomic switch to glucose utilization. Therefore, activation of survival pathways by H11K preemptively triggers the antiapoptotic and metabolic response to ischemia and is sufficient to confer cardioprotection in vivo equally potent to preconditioning.