Differentiation of H9c2 cardiomyoblasts: The role of adenylate cyclase system

Abstract

The adenylate cyclase (AC)/cAMP/cAMP-dependent protein kinase pathway controls many biological phenomena. The molecular mechanisms by which cAMP induces alternative commitment towards differentiation or proliferation are not still completely known. The differentiation of myoblast cell lines into myocytes/myotubes represents a well-established model of skeletal muscle differentiation. We analyzed the AC/cAMP pathway during terminal differentiation of H9c2 myoblasts. When cultured in low-serum containing medium, H9c2 myoblasts exit the cell cycle and differentiate into myocytes/myotubes. A key step of this process is the expression of myogenin, an essential transcription factor for the terminal differentiation into myocytes. During this phenomenon we observed a decrease in both cAMP levels and AC activity, which suggests a functional negative role of cAMP on the differentiation process of H9c2 cells. 8-Br-cAMP and other cAMP-elevating agents, such as forskolin, IBMX, and isoproterenol, negatively affected skeletal muscle differentiation of H9c2 myoblasts. Both AC activity down-regulation and intracellular cAMP reduction were accompanied by significant variations in the levels of membrane proteins belonging to the AC system (AC catalytic subunit, G(alphai-1), G(alphas)). The functional relationship between intracellular cAMP content and protein levels of AC system is discussed.

Cardiomyocyte differentiation: Experience and observations from 2 laboratories

“…This was especially true for cells treated with RA. This observation confirms that although long-term culture in 1% FBS can induce differentiation, (9,18) the addition of RA is necessary to optimally drive the cells in the direction of cardiomyotubes. (20) Investigations into the expression of cardiac specific proteins, specifically cTnT and MCL, revealed a less robust degree of differentiation.…”

Mitochondrial disruption occurs downstream from β-adrenergic overactivation by isoproterenol in differentiated, but not undifferentiated H9c2 cardiomyoblasts: Differential activation of stress and survival pathways

“…Increased basal calcium has been previously described in several models of cell differentiation, including cardiac cells (Fu et al, 2006), mouse embryonic stem cells (ES) (Metzger et al, 1994), human induced pluripotent stem cells (hiPSCs) (Lee et al, 2011) and rat neonatal cardiomyocytes (Gomez et al, 1994). β-receptor activation modulates L-type calcium channels and ryanodine receptors (RyR) (the major sarcoplasmic reticulum calcium release channels in cardiac and skeletal muscles), as well as sarcoplasmic reticulum (SR) Calcium- (Harris, 1977), although differentiated H9c2 myoblasts gradually decreased cAMP and AC activity (Pagano et al, 2004), which may serve as adaptation to increased cytosolic calcium during differentiation. Under normal conditions, calcium stimulates mitochondrial metabolism leading to increased NADH and ATP production (Mildaziene et al, 1996).…”

Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca2+-induced ATP production in cardiac H9c2 myoblasts

“…This cell line possesses cardiac characteristics in the undifferentiated myoblast condition, expressing both cardiac muscle and skeletal muscle-featured proteins. After differentiation, cells form skeletal-type myotubes and lose their cardiac characteristics (39,65,66). Therefore, experiments were performed under the myoblast condition.…”