Cardiomyocytes derived from human embryonic stem (hES) cells potentially offer large numbers of cells to facilitate repair of the infarcted heart. However, this approach has been limited by inefficient differentiation of hES cells into cardiomyocytes, insufficient purity of cardiomyocyte preparations and poor survival of hES cell-derived myocytes after transplantation. Seeking to overcome these challenges, we generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation that relies on activin A and BMP4. We then identified a cocktail of pro-survival factors that limits cardiomyocyte death after transplantation. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls receiving noncardiac hES cell derivatives or vehicle. The ability of hES cell-derived cardiomyocytes to partially remuscularize myocardial infarcts and attenuate heart failure encourages their study under conditions that closely match human disease.

https://courses.washington.edu/conj504/readings/murry_reading2.pdf

Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts

▪ Abstract Eukaryotic translation initiation factor 4F (eIF4F) is a protein complex that mediates recruitment of ribosomes to mRNA. This event is the rate-limiting step for translation under most circumstances and a primary target for translational control. Functions of the constituent proteins of eIF4F include recognition of the mRNA 5′ cap structure (eIF4E), delivery of an RNA helicase to the 5′ region (eIF4A), bridging of the mRNA and the ribosome (eIF4G), and circularization of the mRNA via interaction with poly(A)-binding protein (eIF4G). eIF4 activity is regulated by transcription, phosphorylation, inhibitory proteins, and proteolytic cleavage. Extracellular stimuli evoke changes in phosphorylation that influence eIF4F activity, especially through the phosphoinositide 3-kinase (PI3K) and Ras signaling pathways. Viral infection and cellular stresses also affect eIF4F function. The recent determination of the structure of eIF4E at atomic resolution has provided insight about how translation is initiat...

eIF4 Initiation Factors: Effectors of mRNA Recruitment to Ribosomes and Regulators of Translation

Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.

Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts

http://www.kumc.edu/Documents/msctc/Mangi-Nat%20Med.%20(2003).pdf

Renin is an aspartyl protease essential for the control of blood pressure and was long suspected to have cellular receptors We report the expression cloning of the human renin receptor complementary DNA encoding a 350-amino acid protein with a single transmembrane domain and no homology with any known membrane protein Transfected cells stably expressing the receptor showed renin- and prorenin-specific binding The binding of renin induced a fourfold increase of the catalytic efficiency of angiotensinogen conversion to angiotensin I and induced an intracellular signal with phosphorylation of serine and tyrosine residues associated to an activation of MAP kinases ERK1 and ERK2 High levels of the receptor mRNA are detected in the heart, brain, placenta, and lower levels in the kidney and liver By confocal microscopy the receptor is localized in the mesangium of glomeruli and in the subendothelium of coronary and kidney artery, associated to smooth muscle cells and colocalized with renin The renin receptor is the first described for an aspartyl protease This discovery emphasizes the role of the cell surface in angiotensin II generation and opens new perspectives on the tissue renin-angiotensin system and on renin effects independent of angiotensin II

/pdf/pivotal-role-of-the-renin-prorenin-receptor-in-angiotensin-k0vsk59f6p.pdf

Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin

Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies.

Use of a Biological Peptide Pump to Study Chronic Peptide Hormone Action in Transgenic Mice DIRECT AND INDIRECT EFFECTS OF ANGIOTENSIN II ON THE HEART

The aspartyl protease renin is secreted into the circulation of mammals in 2 forms: the proteolytically processed active form of the enzyme and the precursor form, prorenin. Prorenin has no detectable enzymatic activity in the circulation, but it is the exclusive form of the enzyme produced by several tissues that also produce the other components of the renin enzymatic cascade (renin-angiotensin system). To test whether prorenin might be enzymatically active in these tissues, transgenic mice expressing the human renin substrate (angiotensinogen) exclusively in the pituitary gland were mated to mice expressing either active human renin or prorenin in the same tissue. Measurement of in vivo product formation in pituitary glands of double-transgenic mice revealed that human prorenin was enzymatically active, and Western blot analysis demonstrated that this prorenin was in the precursor form with its prosegment attached. This in vivo enzymatic assay demonstrates for the first time that human prorenin can be activated within tissues by nonproteolytic means, where it could contribute to the activity of a localized renin-angiotensin system.

In Vivo Enzymatic Assay Reveals Catalytic Activity of the Human Renin Precursor in Tissues

The utility of tyrosinase minigene co-injection was evaluated as a visual marker for the generation and breeding of transgenic mice. In an evaluation of 39 transgenic founder animals and 44 transgenic lines five phenotypic patterns of pigmentation were consistently observed, including albino, dark, light, mottled and himalayan. In these studies co-injection of the tyrosinase minigene along with the transgene of interest (TOI) resulted in genomic integration of the two transgenes in 95% of the F0 generation. Co-segregation of transgenes occurred in 94% of doubly transgenic mice in the F1 generation, without dissociation in subsequent generations. All pigmented phenotypes proved useful for distinguishing homozygous from heterozygous F2 animals via backcross trials, while the light, mottled and himalayan phenotypes proved useful in visually discriminating between homozygous and heterozygous F2 animals. In addition, the light, mottled and himalayan phenotypes proved useful in determining segregation patterns of transgenes in the progeny of crosses between separate transgenic lines. Moreover, there appears to be a correlation between intensity of pigmentation and degree of expression of the co-injected TOI. These studies confirm that tyrosinase co-injection is a useful adjunct in transgenic mouse studies and can serve to reduce routine genetic validation of transgenic lines.

Danielle Methot

Papers

Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies.

Use of a Biological Peptide Pump to Study Chronic Peptide Hormone Action in Transgenic Mice DIRECT AND INDIRECT EFFECTS OF ANGIOTENSIN II ON THE HEART

In Vivo Enzymatic Assay Reveals Catalytic Activity of the Human Renin Precursor in Tissues

Evaluation of tyrosinase minigene co-injection as a marker for genetic manipulations in transgenic mice

Tissue Targeting of Angiotensin Peptides