isolation-and-culture-of-neonatal-mouse-cardiomyocytes

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

University of California San Diego 

Cardiac function is regulated by many hormones and neurotransmitters that exert their physiological effects through the activation of G protein-coupled receptors (GPCRs). Identification of new GPCRs that might display a specific pattern of expression within the heart and differentially regulate specific cardiac functions represents an important issue for the development of new drugs. Indeed, highly targeted receptors represent only a small percentage of known GPCRs. Here, we quantified the expression of 395 endoGPCRs (all GPCRs excluding taste and odorant receptors) in male mouse right and left atria and ventricles by using high-throughput real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and focused on the 135 most highly expressed transcripts. No cardiac functional data are available for almost half of these receptors; therefore, linking GPCR expression patterns to cardiac function has allowed us to provide new insights into the possible function of some of these receptors. Indeed, ventricles and atria are both contractile; however, the latter, and especially the right atrium, are central to the generation and regulation of cardiac rhythm. Accordingly, the right atrium exhibited the most specific signature, whereas the majority of GPCRs found in ventricles were evenly expressed in both the right and left chambers. RT-PCR data were confirmed at the protein level for six selected transcripts. Furthermore, we provide new data showing that, as suggested by our repertoire, the metabotropic glutamate receptor 1b is expressed and is functional in ventricular cardiac myocytes. This is the first report describing GPCRs in the four cardiac chambers and may assist in the identification of therapeutic targets.

Identification of potential pharmacological targets by analysis of the comprehensive G protein-coupled receptor repertoire in the four cardiac chambers.

Thymosin beta 4 (TÎ²4) is a 43-amino acid factor encoded by an X-linked gene. Recent studies have suggested that TÎ²4 is a key factor in cardiac development, growth, disease, epicardial integrity, and blood vessel formation. Cardiac-specific short hairpin (sh)RNA knockdown of tÎ²4 has been reported to result in embryonic lethality at E14.5-16.5, with severe cardiac and angiogenic defects. However, this shRNA tÎ²4-knockdown model did not completely abrogate TÎ²4 expression. To completely ablate TÎ²4 and to rule out the possibility of off-target effects associated with shRNA gene silencing, further studies of global or cardiac-specific knockouts are critical.

Thymosin beta 4 is dispensable for murine cardiac development and function.

Myofibroblasts produce the fibrous scar in hepatic fibrosis. In the carbon tetrachloride (CCl(4)) model of liver fibrosis, quiescent hepatic stellate cells (HSC) are activated to become myofibroblasts. When the underlying etiological agent is removed, clinical and experimental fibrosis undergoes a remarkable regression with complete disappearance of these myofibroblasts. Although some myofibroblasts apoptose, it is unknown whether other myofibroblasts may revert to an inactive phenotype during regression of fibrosis. We elucidated the fate of HSCs/myofibroblasts during recovery from CCl(4)- and alcohol-induced liver fibrosis using Cre-LoxP-based genetic labeling of myofibroblasts. Here we demonstrate that half of the myofibroblasts escape apoptosis during regression of liver fibrosis, down-regulate fibrogenic genes, and acquire a phenotype similar to, but distinct from, quiescent HSCs in their ability to more rapidly reactivate into myofibroblasts in response to fibrogenic stimuli and strongly contribute to liver fibrosis. Inactivation of HSCs was associated with up-regulation of the anti-apoptotic genes Hspa1a/b, which participate in the survival of HSCs in culture and in vivo.

Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis.

Rossdeutsch et al describe a requirement for thymosin Î²4 (TÎ²4) in vascular development. Impaired mural cell migration, differentiation, partial embryonic lethality, and hemorrhaging were observed after analysis of 2 lines of mice, one of which was germline null for TÎ²4 and another in which TÎ²4 was knocked down by endothelial-specific expression of TÎ²4 short hairpin RNA. These data are in direct contrast to our published global and cardiac-specific TÎ²4-knockout lines. Thus, the role of TÎ²4 needs to be clarified to understand its importance in cardiovascular development.

Thomas Moore-Morris

Division of Cardiology,
School of Medicine,
Division of Cardiology, School of Medicine

University of California San Diego

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Thomas Moore-Morris

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Cardiology,School of Medicine,Division of Cardiology, School of Medicine

University of California San Diego

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Division of Cardiology,
School of Medicine,
Division of Cardiology, School of Medicine