Kenneth R. Chien

PPARγ Is Required for Placental, Cardiac, and Adipose Tissue Development

The nuclear hormone receptor PPAR gamma promotes adipogenesis and macrophage differentiation and is a primary pharmacological target in the treatment of type II diabetes. Here, we show that PPAR gamma gene knockout results in two independent lethal phases. Initially, PPAR gamma deficiency interferes with terminal differentiation of the trophoblast and placental vascularization, leading to severe myocardial thinning and death by E10.0. Supplementing PPAR gamma null embryos with wild-type placentas via aggregation with tetraploid embryos corrects the cardiac defect, implicating a previously unrecognized dependence of the developing heart on a functional placenta. A tetraploid-rescued mutant surviving to term exhibited another lethal combination of pathologies, including lipodystrophy and multiple hemorrhages. These findings both confirm and expand the current known spectrum of physiological functions regulated by PPAR gamma.

Multipotent Embryonic Isl1+ Progenitor Cells Lead to Cardiac, Smooth Muscle, and Endothelial Cell Diversification

Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1(+) precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1(+) cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1(+)/Nkx2.5(+)/flk1(+) defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1(+) cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.

Cardiac Muscle Cell Hypertrophy and Apoptosis Induced by Distinct Members of the p38 Mitogen-activated Protein Kinase Family

p38 mitogen-activated protein (MAP) kinase activities were significantly increased in mouse hearts after chronic transverse aortic constriction, coincident with the onset of ventricular hypertrophy. Infection of cardiomyocytes with adenoviral vectors expressing upstream activators for the p38 kinases, activated mutants of MAP kinase kinase 3b(E) (MKK3bE) and MAP kinase kinase 6b(E) (MKK6bE), elicited characteristic hypertrophic responses, including an increase in cell size, enhanced sarcomeric organization, and elevated atrial natriuretic factor expression. Overexpression of the activated MKK3bE in cardiomyocytes also led to an increase in apoptosis. The hypertrophic response was enhanced by co-infection of an adenoviral vector expressing wild type p38β, and was suppressed by the p38β dominant negative mutant. In contrast, the MKK3bE-induced cell death was increased by co-infection of an adenovirus expressing wild type p38α, and was suppressed by the dominant negative p38α mutant. This provides the first evidence in any cell system for divergent physiological functions for different members of the p38 MAP kinase family. The direct involvement of p38 pathways in cardiac hypertrophy and apoptosis suggests a significant role for p38 signaling in the pathophysiology of heart failure.

MLP-Deficient Mice Exhibit a Disruption of Cardiac Cytoarchitectural Organization, Dilated Cardiomyopathy, and Heart Failure

MLP is a LIM-only protein of terminally differentiated striated muscle cells, where it accumulates at actin-based structures involved in cytoarchitecture organization. To assess its role in muscle differentiation, we disrupted the MLP gene in mice. MLP (-/-) mice developed dilated cardiomyopathy with hypertrophy and heart failure after birth. Ultrastructural analysis revealed dramatic disruption of cardiomyocyte cytoarchitecture. At birth, these hearts were not hypertrophic, but already abnormally soft, with cell-autonomous and MLP-sensitive alterations in cytoarchitecture. Thus, MLP promotes proper cardiomyocyte cytoarchitecture, whose perturbation can lead to dilated cardiomyopathy. In vivo analysis revealed that MLP-deficient mice reproduce the morphological and clinical picture of dilated cardiomyopathy and heart failure in humans, providing the first model for this condition in a genetically manipulatable organism.

ErbB2 is essential in the prevention of dilated cardiomyopathy

Amplification of the gene encoding the ErbB2 (Her2/neu) receptor tyrosine kinase is critical for the progression of several forms of breast cancer. In a large-scale clinical trial, treatment with Herceptin (trastuzumab), a humanized blocking antibody against ErbB2, led to marked improvement in survival. However, cardiomyopathy was uncovered as a mitigating side effect, thereby suggesting an important role for ErbB2 signaling as a modifier of human heart failure. To investigate the physiological role of ErbB2 signaling in the adult heart, we generated mice with a ventricular-restricted deletion of Erbb2. These ErbB2-deficient conditional mutant mice were viable and displayed no overt phenotype. However, physiological analysis revealed the onset of multiple independent parameters of dilated cardiomyopathy, including chamber dilation, wall thinning and decreased contractility. Additionally, cardiomyocytes isolated from these conditional mutants were more susceptible to anthracycline toxicity. ErbB2 signaling in cardiomyocytes is therefore essential for the prevention of dilated cardiomyopathy.

Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart

The heart is formed from cardiogenic progenitors expressing the transcription factors Nkx2-5 and Isl1 (refs 1 and 2). These multipotent progenitors give rise to cardiomyocyte, smooth muscle and endothelial cells, the major lineages of the mature heart. Here we identify a novel cardiogenic precursor marked by expression of the transcription factor Wt1 and located within the epicardium—an epithelial sheet overlying the heart. During normal murine heart development, a subset of these Wt1+ precursors differentiated into fully functional cardiomyocytes. Wt1+ proepicardial cells arose from progenitors that express Nkx2-5 and Isl1, suggesting that they share a developmental origin with multipotent Nkx2-5+ and Isl1+ progenitors. These results identify Wt1+ epicardial cells as previously unrecognized cardiomyocyte progenitors, and lay the foundation for future efforts to harness the cardiogenic potential of these progenitors for cardiac regeneration and repair.

The Cardiac Mechanical Stretch Sensor Machinery Involves a Z Disc Complex that Is Defective in a Subset of Human Dilated Cardiomyopathy

Muscle cells respond to mechanical stretch stimuli by triggering downstream signals for myocyte growth and survival. The molecular components of the muscle stretch sensor are unknown, and their role in muscle disease is unclear. Here, we present biophysical/biochemical studies in muscle LIM protein (MLP) deficient cardiac muscle that support a selective role for this Z disc protein in mechanical stretch sensing. MLP interacts with and colocalizes with telethonin (T-cap), a titin interacting protein. Further, a human MLP mutation (W4R) associated with dilated cardiomyopathy (DCM) results in a marked defect in T-cap interaction/localization. We propose that a Z disc MLP/T-cap complex is a key component of the in vivo cardiomyocyte stretch sensor machinery, and that defects in the complex can lead to human DCM and associated heart failure.

Loss of a gp130 Cardiac Muscle Cell Survival Pathway Is a Critical Event in the Onset of Heart Failure during Biomechanical Stress

Biomechanical stress is a major stimulus for cardiac hypertrophy and the transition to heart failure. By generating mice that harbor a ventricular restricted knockout of the gp130 cytokine receptor via Cre-IoxP-mediated recombination, we demonstrate a critical role for a gp130-dependent myocyte survival pathway in the transition to heart failure. Such conditional mutant mice have normal cardiac structure and function, but during aortic pressure overload, these mice display rapid onset of dilated cardiomyopathy and massive induction of myocyte apoptosis versus the control mice that exhibit compensatory hypertrophy. Thus, cardiac myocyte apoptosis is a critical point in the transition between compensatory cardiac hypertrophy and heart failure. gp130-dependent cytokines may represent a novel therapeutic strategy for preventing in vivo heart failure.

Marked differences in differentiation propensity among human embryonic stem cell lines

The differentiation potential of 17 human embryonic stem (hES) cell lines was compared. Some lines exhibit a marked propensity to differentiate into specific lineages, often with >100-fold differences in lineage-specific gene expression. For example, HUES 8 is best for pancreatic differentiation and HUES 3 for cardiomyocyte generation. These non-trivial differences in developmental potential among hES cell lines point to the importance of screening and deriving lines for lineage-specific differentiation.

IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages

Whereas interleukin-6 (IL-6) is a proinflammatory cytokine, IL-10 is an anti-inflammatory cytokine. Although signal transducer and activator of transcription 3 (STAT3) is essential for the function of both IL-6 and IL-10, it is unclear how these two cytokines have such opposing functions. Here we show that suppressor of cytokine signaling 3 (SOCS3) is a key regulator of the divergent action of these two cytokines. In macrophages lacking the Socs3 gene or carrying a mutation of the SOCS3-binding site in gp130, the lipopolysaccharide-induced production of tumor necrosis factor (TNF) and IL-12 is suppressed by both IL-10 and IL-6. SOCS3 specifically prevents activation of STAT3 by IL-6 but not IL-10. Taken together, these data indicate that SOCS3 selectively blocks signaling by IL-6, thereby preventing its ability to inhibit LPS signaling.