scholarly journals Generating Primary Cultures of Murine Cardiac Myocytes and Cardiac Fibroblasts to Study Viral Myocarditis

2015 ◽  
pp. 1-16 ◽  
Author(s):  
Barbara Sherry
Keyword(s):  
Cardiac Myocytes ◽  
Cardiac Fibroblasts ◽  
Primary Cultures ◽  
Viral Myocarditis

scholarly journals Basal and Reovirus-Induced Beta Interferon (IFN-β) and IFN-β-Stimulated Gene Expression Are Cell Type Specific in the Cardiac Protective Response

2005 ◽  
Vol 79 (5) ◽  
pp. 2979-2987 ◽  
Author(s):  
Michael J. Stewart ◽  
Kathleen Smoak ◽  
Mary Ann Blum ◽  
Barbara Sherry
Keyword(s):  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Cardiac Fibroblasts ◽  
Viral Myocarditis ◽  
Cell Types ◽  
Protective Role ◽  
Cell Type ◽  
Cardiac Damage ◽  
Beta Interferon ◽  
Reovirus Replication

ABSTRACT Viral myocarditis is an important human disease, with a wide variety of viruses implicated. Cardiac myocytes are not replenished yet are critical for host survival and thus may have a unique response to infection. Previously, we determined that the extent of reovirus induction of beta interferon (IFN-β) and IFN-β-mediated protection in primary cardiac myocyte cultures was inversely correlated with the extent of reovirus-induced cardiac damage in a mouse model. Surprisingly, and in contrast, the IFN-β response did not determine reovirus replication in skeletal muscle cells. Here we compared the IFN-β response in cardiac myocytes to that in primary cardiac fibroblast cultures, a readily replenished cardiac cell type. We compared basal and reovirus-induced expression of IFN-β, IRF-7 (an interferon-stimulated gene [ISG] that further induces IFN-β), and another ISG (561) in the two cell types by using real-time reverse transcription-PCR. Basal IFN-β, IRF-7, and 561 expression was higher in cardiac myocytes than in cardiac fibroblasts. Reovirus T3D induced greater expression of IFN-β in cardiac myocytes than in cardiac fibroblasts but equivalent expression of IRF-7 and 561 in the two cell types (though fold induction for IRF-7 and 561 was higher in fibroblasts than in myocytes because of the differences in basal expression). Interestingly, while reovirus replicated to equivalent titers in cardiac myocytes and cardiac fibroblasts, removal of IFN-β resulted in 10-fold-greater reovirus replication in the fibroblasts than in the myocytes. Together the data suggest that the IFN-β response controls reovirus replication equivalently in the two cell types. In the absence of reovirus-induced IFN-β, however, reovirus replicates to higher titers in cardiac fibroblasts than in cardiac myocytes, suggesting that the higher basal IFN-β and ISG expression in myocytes may play an important protective role.

scholarly journals Basal Expression Levels of IFNAR and Jak-STAT Components Are Determinants of Cell-Type-Specific Differences in Cardiac Antiviral Responses

2007 ◽  
Vol 81 (24) ◽  
pp. 13668-13680 ◽  
Author(s):  
Jennifer Zurney ◽  
Kristina E. Howard ◽  
Barbara Sherry
Keyword(s):  
Cardiac Myocytes ◽  
Cardiac Fibroblasts ◽  
Viral Myocarditis ◽  
Cell Types ◽  
Cell Type ◽  
Organ Protection ◽  
Integrated Network ◽  
Basal Expression ◽  
Cell Type Specific ◽  
Β Receptor

ABSTRACT Viral myocarditis is an important human disease, and reovirus-induced murine myocarditis provides an excellent model system for study. Cardiac myocytes, like neurons in the central nervous system, are not replenished, yet there is no cardiac protective equivalent to the blood-brain barrier. Thus, cardiac myocytes may have evolved a unique antiviral response relative to readily replenished cell types, such as cardiac fibroblasts. Our previous comparisons of these two cell types revealed a conundrum: reovirus T3D induces more beta-interferon (IFN-β) mRNA in cardiac myocytes, yet there is a greater induction of IFN-stimulated genes (ISGs) in cardiac fibroblasts. Here, we investigated possible underlying molecular determinants. We found that greater basal expression of IFN-β in cardiac myocytes results in greater basal activated nuclear STAT1 and STAT2 and greater basal ISG mRNA expression and provides greater basal antiviral protection relative to cardiac fibroblasts. Conversely, cardiac fibroblasts express greater basal IFN-α/β receptor 1 (IFNAR1) and greater basal cytoplasmic Jak1, Tyk2, STAT2, and IRF9, leading to a greater increase in reovirus T3D- or IFN-induced nuclear activated STAT1 and STAT2 and greater induction of ISGs for a greater IFN-induced antiviral protection relative to cardiac myocytes. Our results suggest that high basal IFN-β expression in cardiac myocytes prearms this vulnerable, nonreplenishable cell type, while high basal expression of IFNAR1 and latent Jak-STAT components in adjacent cardiac fibroblasts renders these cells more responsive to IFN and prevents them from inadvertently serving as a reservoir for viral replication and spread to cardiac myocytes. These studies provide the first indication of an integrated network of cell-type-specific innate immune components for organ protection.

scholarly journals Changes in the fluctuation of the contraction rhythm of spontaneously beating cardiac myocytes in cultures with and without cardiac fibroblasts

Biosystems ◽  
2007 ◽  
Vol 90 (3) ◽  
pp. 707-715 ◽  
Author(s):  
Takeru Hachiro ◽  
Koichi Kawahara ◽  
Rie Sato ◽  
Yoshiko Yamauchi ◽  
Daisuke Matsuyama
Keyword(s):  
Cardiac Myocytes ◽  
Cardiac Fibroblasts ◽  
Contraction Rhythm

scholarly journals Paracrine effects of hypoxic fibroblast-derived factors on the MPT-ROS threshold and viability of adult rat cardiac myocytes

2008 ◽  
Vol 294 (6) ◽  
pp. H2653-H2658 ◽  
Author(s):  
K. Shivakumar ◽  
S. J. Sollott ◽  
M. Sangeetha ◽  
S. Sapna ◽  
B. Ziman ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Conditioned Medium ◽  
Cardiac Myocytes ◽  
Cytokine Production ◽  
Mitochondrial Permeability Transition ◽  
Cardiac Fibroblasts ◽  
Ischemia Reperfusion ◽  
Oxidant Stress ◽  
Cardiac Cells ◽  
Tnf Α

Cardiac fibroblasts contribute to multiple aspects of myocardial function and pathophysiology. The pathogenetic relevance of cytokine production by these cells under hypoxia, however, remains unexplored. With the use of an in vitro cell culture model, this study evaluated cytokine production by hypoxic cardiac fibroblasts and examined two distinct effects of hypoxic fibroblast-conditioned medium (HFCM) on cardiac myocytes and fibroblasts. Hypoxia caused a marked increase in the production of tumor necrosis factor (TNF)-α by cardiac fibroblasts. HFCM significantly enhanced the susceptibility of cardiac myocytes to reactive oxygen species (ROS)-induced mitochondrial permeability transition (MPT), determined by high-precision confocal line-scan imaging following controlled, photoexcitation-induced ROS production within individual mitochondria. Furthermore, exposure of cardiac myocytes to HFCM for 5 h led to loss of viability, as evidenced by change in morphology and annexin staining. HFCM also decreased DNA synthesis in cardiac fibroblasts. Normoxic fibroblast-conditioned medium spiked with TNF-α at 200 pg/ml, a concentration comparable to that in HFCM, promoted loss of myocyte viability and decreased DNA synthesis in cardiac fibroblasts. These effects of HFCM are similar to the reported effects of hypoxia per se on these cell types, showing that hypoxic fibroblast-derived factors may amplify the distinct effects of hypoxia on cardiac cells. Importantly, because both hypoxia and oxidant stress prevail in a setting of ischemia and reperfusion, the effects of soluble factors from hypoxic fibroblasts on the MPT-ROS threshold and viability of myocytes may represent a novel paracrine mechanism that could exacerbate ischemia-reperfusion injury to cardiomyocytes.

CTRP15 derived from cardiac myocytes attenuates TGFβ1-induced fibrotic response in cardiac fibroblasts

2020 ◽  
Vol 34 (5) ◽  
pp. 591-604
Author(s):  
Qian Zhao ◽  
Cheng-Lin Zhang ◽  
Ruo-Lan Xiang ◽  
Li-Ling Wu ◽  
Li Li
Keyword(s):  
Cardiac Myocytes ◽  
Cardiac Fibroblasts ◽  
Fibrotic Response

scholarly journals Cardiac Fibroblasts Aggravate Viral Myocarditis: Cell Specific Coxsackievirus B3 Replication

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Diana Lindner ◽  
Jia Li ◽  
Konstantinos Savvatis ◽  
Karin Klingel ◽  
Stefan Blankenberg ◽  
...  
Keyword(s):  
Viral Infection ◽  
Virus Replication ◽  
Cardiac Tissue ◽  
Cardiac Fibroblasts ◽  
Viral Myocarditis ◽  
Fold Increase ◽  
Coxsackievirus B3 ◽  
Virus Concentration ◽  
Cardiac Inflammation ◽  
Viral Copy Number

Myocarditis is an inflammatory disease caused by viral infection. Different subpopulations of leukocytes enter the cardiac tissue and lead to severe cardiac inflammation associated with myocyte loss and remodeling. Here, we study possible cell sources for viral replication using three compartments of the heart: fibroblasts, cardiomyocytes, and macrophages. We infected C57BL/6j mice with Coxsackievirus B3 (CVB3) and detected increased gene expression of anti-inflammatory and antiviral cytokines in the heart. Subsequently, we infected cardiac fibroblasts, cardiomyocytes, and macrophages with CVB3. Due to viral infection, the expression of TNF-α, IL-6, MCP-1, and IFN-βwas significantly increased in cardiac fibroblasts compared to cardiomyocytes or macrophages. We found that in addition to cardiomyocytes cardiac fibroblasts were infected by CVB3 and displayed a higher virus replication (132-fold increase) compared to cardiomyocytes (14-fold increase) between 6 and 24 hours after infection. At higher virus concentrations, macrophages are able to reduce the viral copy number. At low virus concentration a persistent virus infection was determined. Therefore, we suggest that cardiac fibroblasts play an important role in the pathology of CVB3-induced myocarditis and are another important contributor of virus replication aggravating myocarditis.

scholarly journals Moderate Calcium Channel Dysfunction in Adult Mice with Inducible Cardiomyocyte-specific Excision of the cacnb2 Gene

2011 ◽  
Vol 286 (18) ◽  
pp. 15875-15882 ◽  
Author(s):  
Marcel Meissner ◽  
Petra Weissgerber ◽  
Juan E. Camacho Londoño ◽  
Jean Prenen ◽  
Sabine Link ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Heart Function ◽  
Cardiac Fibroblasts ◽  
Calcium Currents ◽  
Protein Fractions ◽  
Protein Levels ◽  
Adult Mice ◽  
Embryonic Cardiomyocytes ◽  
The Difference

The major L-type voltage-gated calcium channels in heart consist of an α1C (CaV1.2) subunit usually associated with an auxiliary β subunit (CaVβ2). In embryonic cardiomyocytes, both the complete and the cardiac myocyte-specific null mutant of CaVβ2 resulted in reduction of L-type calcium currents by up to 75%, compromising heart function and causing defective remodeling of intra- and extra-embryonic blood vessels followed by embryonic death. Here we conditionally excised the CaVβ2 gene (cacnb2) specifically in cardiac myocytes of adult mice (KO). Upon gene deletion, CaVβ2 protein expression declined by >96% in isolated cardiac myocytes and by >74% in protein fractions from heart. These latter protein fractions include CaVβ2 proteins expressed in cardiac fibroblasts. Surprisingly, mice did not show any obvious impairment, although cacnb2 excision was not compensated by expression of other CaVβ proteins or changes of CaV1.2 protein levels. Calcium currents were still dihydropyridine-sensitive, but current density at 0 mV was reduced by <29%. The voltage for half-maximal activation was slightly shifted to more depolarized potentials in KO cardiomyocytes when compared with control cells, but the difference was not significant. In summary, CaVβ2 appears to be a much stronger modulator of L-type calcium currents in embryonic than in adult cardiomyocytes. Although essential for embryonic survival, CaVβ2 down-regulation in cardiomyocytes is well tolerated by the adult mice.

scholarly journals Urocortin increases the expression of heat shock protein 90 in rat cardiac myocytes in a MEK1/2-dependent manner

2002 ◽  
Vol 172 (2) ◽  
pp. 283-293 ◽  
Author(s):  
BK Brar ◽  
J Railson ◽  
A Stephanou ◽  
RA Knight ◽  
DS Latchman
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Cardiac Myocytes ◽  
Primary Cultures ◽  
Heat Shock Protein 90 ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Maximal Increase ◽  
Mitogen Activated Protein ◽  
Hsp90 Protein

We have previously demonstrated that urocortin protects cultured cardiac myocytes from ischaemic and reoxygenation injury and decreases the infarct size in the rat heart exposed to regional ischaemia and reperfusion. Urocortin-mediated cardioprotection is via activation of the mitogen-activated protein kinase (MAP kinase, MEK1/2) pathway. In addition, it is well documented that heat shock protein (hsp) 70 and hsp90 are cardioprotective against lethal stress. In this study we show, for the first time, that urocortin induces the expression of hsp90 but not hsp70 in primary cultures of rat neonatal cardiac myocytes. Levels of hsp90 protein increase by 1.5-fold over untreated cells within 10 min of urocortin treatment and are sustained for 24 h with a maximal increase of 2.5-fold at 60 min (P<0.05 at all time points). The increase in hsp90 expression by urocortin was not inhibited by actinomycin D, and urocortin failed to increase hsp90 promoter activity. Urocortin induction of hsp90 was inhibited by the MEK1/2 inhibitor PD98059 (P<0.001) and by cycloheximide, and both inhibitors abrogate urocortin-mediated cardioprotection (P<0.05 for cycloheximide, P<0.001 for PD98059). Hence, MEK1/2 and protein synthesis are involved in the cardioprotective effect of urocortin against hypoxic-mediated cell death, possibly due to an increase in expression of hsp90 protein. This is the first report of heat shock protein induction by urocortin or any other member of the corticotrophin-releasing hormone family.

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