Cell Cycle Re-Entry and Mitochondrial Defects in Myc-Mediated Hypertrophic Cardiomyopathy and Heart Failure

While considerable evidence supports the causal relationship between increases in c-Myc (Myc) and cardiomyopathy as a part of a “fetal re-expression” pattern, the functional role of Myc in mechanisms of cardiomyopathy remains unclear. To address this, we developed a bitransgenic mouse that inducibly expresses Myc under the control of the cardiomyocyte-specific MHC promoter. In adult mice the induction of Myc expression in cardiomyocytes in the heart led to the development of severe hypertrophic cardiomyopathy followed by ventricular dysfunction and ultimately death from congestive heart failure. Mechanistically, following Myc activation, cell cycle markers and other indices of DNA replication were significantly increased suggesting that cell cycle-related events might be a primary mechanism of cardiac dysfunction. Furthermore, pathological alterations at the cellular level included alterations in mitochondrial function with dysregulation of mitochondrial biogenesis and defects in electron transport chain complexes I and III. These data are consistent with the known role of Myc in several different pathways including cell cycle activation, mitochondrial proliferation, and apoptosis, and indicate that Myc activation in cardiomyocytes is an important regulator of downstream pathological sequelae. Moreover, our findings indicate that the induction of Myc in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure, and that sustained induction of Myc, leading to cell cycle re-entry in adult cardiomyocytes, represents a maladaptive response for the mature heart

Insulin-like growth factors and their potential role in cardiac epigenetics

Cardiovascular disease (CVD) constitutes a major public health threat worldwide, accounting for 17.3 million deaths annually. Heart disease and stroke account for the majority of healthcare costs in the developed world. While much has been accomplished in understanding the pathophysiology, molecular biology and genetics underlying the diagnosis and treatment of CVD, we know less about the role of epigenetics and their molecular determinants. The impact of environmental changes and epigenetics in CVD is now emerging as critically important in understanding the origin of disease and the development of new therapeutic approaches to prevention and treatment. This review focuses on the emerging role of epigenetics mediated by insulin like-growth factors-I and -II in major CVDs such as heart failure, cardiac hypertrophy and diabetes

Knockdown of subunit 3 of the COP9 signalosome inhibits C2C12 myoblast differentiation via NF-KappaB signaling pathway

Abstract Background The COP9 signalosome (CSN) is a conserved protein complex composed of 8 subunits designated CSN1-CSN8. CSN3 represents the third subunit of the CSN and maintains the integrity of the complex. CSN3 binds to the striated muscle-specific β1D integrin tail, and its subcellular localization is altered in differentiated skeletal muscle cells. However, the role of CSN3 in skeletal muscle differentiation is unknown. The main goal of this study was to identify whether CSN3 participates in myoblast differentiation and the signalling mechanisms involved using C2C12 cells as a skeletal muscle cell model. Methods Small-hairpin (shRNA) was used to knockdown CSN3 in C2C12 cells. Differentiation was evaluated by immunostaining and confocal microscopy. Markers of differentiation, NF-κB signaling and CSN subunits expression, were assessed by immunoblotting and/or immunostaining. Cell proliferation was analysed by cell counting, flow cytometry and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data were analyzed by one or two-way analysis of variance (ANOVA) followed by post-hoc testing. Results Transduction of C2C12 cells with two distinct CSN3 shRNAs led to the production of two cells lines expressing 7% of CSN3 protein (shCSN3-Low) and 43% of CSN3 protein (CSN3-Med) compared to controls. Knockdown of CSN3 was accompanied by destabilization of several CSN subunits and increased nuclear NF-κB localization. shCSN3-Med cells expressed less myogenin and formed shorter and thinner myotubes. In contrast, the shCSN3-Low cells expressed higher levels of myogenin prior and during the differentiation and remained mononucleated throughout the differentiation period. Both CSN3 knockdown cell lines failed to express sarcomeric myosin heavy chain (MHC) protein during differentiation. The fusion index was significantly higher in control cells than in shCSN3-Med cells, whereas shCSN3-Low cells showed no cell fusion. Interestingly, CSN3 knockdown cells exhibited a significantly slower growth rate relative to the control cells. Cell cycle analysis revealed that CSN3 knockdowns delayed in S phase and had increased levels of nuclear p21/Cip1 and p27/Kip1. Conclusions This study clarifies the first step toward unrevealing the CSN3/CSN-mediated pathways that controls C2C12 differentiation and proliferation. Further in vivo characterization of CSN/CSN3 may lead to the discovery of novel therapeutic target of skeletal muscle diseases such as muscular dystrophies

Influence of the extracellular matrix and integrins on volume-sensitive osmolyte anion channels in C2C12 myoblasts

The purpose of this study was to determine whether extracellular matrix (ECM) composition through integrin receptors modulated the volume-sensitive osmolyte anion channels (VSOACs) in skeletal muscle-derived C2C12 cells. Cl− currents were recorded in whole cell voltage-clamped cells grown on laminin (LM), fibronectin (FN), or in the absence of a defined ECM (NM). Basal membrane currents recorded in isotonic media (300 mosmol/kg) were larger in cells grown on FN (3.8-fold at +100 mV) or LM (8.8-fold at +100 mV) when compared with NM. VSOAC currents activated by cell exposure to hypotonic solution were larger in cells grown on LM (1.72-fold at +100 mV) or FN (1.75-fold at +100 mV) compared with NM. Additionally, the kinetics of VSOAC activation was ≈27% quicker on FN and LM. These currents were tamoxifen sensitive, displayed outward rectification, reversed at the equilibrium potential of Cl− and inactivated at potentials >+60 mV. Specific knockdown of β1-integrin by short hairpin RNA interference strongly inhibited the VSOAC Cl− currents in cells plated on FN. In conclusion, ECM composition and integrins profoundly influence the biophysical properties and mechanisms of onset of VSOACs

Doxycycline inducible expression of SERCA2a improves calcium handling and reverts cardiac dysfunction in pressure overload-induced cardiac hypertrophy

Delayed cardiac relaxation in failing hearts has been attributed to reduced activity and/or expression of sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a). Although constitutive overexpression of SERCA2a has proven effective in preventing cardiac dysfunction, it is unclear whether increasing SERCA2a expression in hearts with preexisting hypertrophy will be therapeutic. To test this hypothesis, we generated a binary transgenic (BTG) system that allows tetracycline-inducible, cardiac-specific SERCA2a expression. In this system (tet-on SERCA2a), a FLAG-tagged SERCA2a transgene is expressed in the presence of doxycycline (Dox) but not in the absence of Dox (2.3-fold more mRNA, 45% more SERCA2a protein). Calcium transients measured in isolated cardiac myocytes from nonbanded Dox-treated BTG mice showed an accelerated calcium decline and an increased systolic Ca2+ peak. Sarcoplasmic reticulum (SR) calcium loading was increased by 45% in BTG mice. In the presence of pressure overload (aortic banding), echocardiographic analysis revealed that expression of SERCA2a-FLAG caused an improvement in fractional shortening. SERCA2a-FLAG expression alleviated the resultant cardiac dysfunction. This was illustrated by an increase in the rate of decline of the calcium transient. Cell shortening and SR calcium loading were also improved in cardiac myocytes isolated from banded BTG mice after SERCA2a overexpression. In conclusion, we generated a novel transgenic mouse that conditionally overexpresses SERCA2a. This model is suitable for both long- and short-term studies of the effects of controlled SERCA2a expression on cardiac function. In addition, inducible overexpression of SERCA2a improved cardiac function and calcium handling in mice with established contractile dysfunction