Ankrd1/CARP, Ankrd2/Arpp, and Ankrd23/DARP belong to a family of stress inducible ankyrin repeat proteins expressed in striated muscle (MARPs). The MARPs are homologous in structure and localized in the nucleus where they negatively regulate gene expression as well as in the sarcomeric I-band, where they are thought to be involved in mechanosensing. Together with their strong induction during cardiac disease and the identification of causative Ankrd1 gene mutations in cardiomyopathy patients, this suggests their important roles in cardiac development, function, and disease. To determine the functional role of MARPs in vivo, we studied knockout (KO) mice of each of the three family members. Single KO mice were viable and had no apparent cardiac phenotype. We therefore hypothesized that the three highly homologous MARP proteins may have redundant functions in the heart and studied double and triple MARP KO mice. Unexpectedly, MARP triple KO mice were viable and had normal cardiac function both at basal levels and in response to mechanical pressure overload induced by transverse aortic constriction as assessed by echocardiography and hemodynamic studies. Thus, CARP, Ankrd2, and DARP are not essential for normal cardiac development and function at basal conditions and in response to mechanical pressure overload.

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.

It has long been held as scientific fact that soon after birth, cardiomyocytes cease dividing, thus explaining the limited restoration of cardiac function after a heart attack. Recent demonstrations of cardiac myocyte differentiation observed in vitro or after in vivo transplantation of adult stem cells from blood, fat, skeletal muscle, or heart have challenged this view. Analysis of these studies has been complicated by the large disparity in the magnitude of effects seen by different groups and obscured by the recently appreciated process of in vivo stem-cell fusion. We now show a novel population of nonsatellite cells in adult murine skeletal muscle that progress under standard primary cell-culture conditions to autonomously beating cardiomyocytes. Their differentiation into beating cardiomyocytes is characterized here by video microscopy, confocal-detected calcium transients, electron microscopy, immunofluorescent cardiac-specific markers, and single-cell patch recordings of cardiac action potentials. Within 2 d after tail-vein injection of these marked cells into a mouse model of acute infarction, the marked cells are visible in the heart. By 6 d they begin to differentiate without fusing to recipient cardiac cells. Three months later, the tagged cells are visible as striated heart muscle restricted to the region of the cardiac infarct.

The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate T-tubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2) to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC) coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling, and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubule-based Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient, indicating that Cav1.2 targeting to BIN1 is functionally important to cardiac calcium signaling. We have identified that membrane-associated BIN1 not only induces membrane curvature but can direct specific antegrade delivery of microtubule-transported membrane proteins. Furthermore, this paradigm provides a microtubule and BIN1-dependent mechanism of Cav1.2 delivery to T-tubules. This novel Cav1.2 trafficking pathway should serve as an important regulatory aspect of EC coupling, affecting cardiac contractility in mammalian hearts.