Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression

Kabaeva, Zhyldyz; Zhao, Mei; Michele, Daniel E.

doi:10.1152/ajpheart.01144.2007

Cited by 83 publications

(79 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Isolation of cardiomyocytes from adult mouse hearts is known to be much more technically demanding than isolation of adult rat cardiomyocytes (13). Using the protocol described here, we are able to reproducibly obtain sufficient numbers of viable cells for contractility and other functional studies (data not shown).…”

Section: Discussionmentioning

confidence: 89%

A dominantly negative mutation in cardiac troponin I at the interface with troponin T causes early remodeling in ventricular cardiomyocytes

Wei

Jin

2014

American Journal of Physiology-Cell Physiology

View full text Add to dashboard Cite

Wei H, Jin JP. A dominantly negative mutation in cardiac troponin I at the interface with troponin T causes early remodeling in ventricular cardiomyocytes. Am J Physiol Cell Physiol 307: C338-C348, 2014. First published June 4, 2014; doi:10.1152/ajpcell.00053.2014.-We previously reported a point mutation substituting Cys for Arg 111 in the highly conserved troponin T (TnT)-contacting helix of cardiac troponin I (cTnI) in wild turkey hearts (Biesiadecki et al. J Biol Chem 279: 13825-13832, 2004). This dominantly negative TnI-TnT interface mutation decreases the binding affinity of cTnI for TnT, impairs diastolic function, and blunts the ␤-adrenergic response of cardiac muscle (Wei et al. J Biol Chem 285: 27806 -27816, 2010). Here we further investigate cellular phenotypes of transgenic mouse cardiomyocytes expressing the equivalent mutation cTnI-K118C. Functional studies were performed on single adult cardiomyocytes after recovery in short-term culture from isolation stress. The amplitude of contraction and the velocities of shortening and relengthening were lower in cTnI-K118C cardiomyocytes than wild-type controls. The intracellular Ca 2ϩ transient was slower in cTnI-K118C cardiomyocytes than wild-type cells. cTnI-K118C cardiomyocytes also showed a weaker ␤-adrenergic response. The resting length of cTnI-K118C cardiomyocytes was significantly greater than that of age-matched wild-type cells, with no difference in cell width. The resting sarcomere was not longer, but slightly shorter, in cTnI-K118C cardiomyocytes than wild-type cells, indicating longitudinal addition of sarcomeres. More tri-and quadrinuclei cardiomyocytes were found in TnI-K118C than wild-type hearts, suggesting increased nuclear divisions. Whole-genome mRNA array and Western blots detected an increased expression of leukemia inhibitory factor receptor-␤ in the hearts of 2-mo-old cTnI-K118C mice, suggesting a signaling pathway responsible for the potent effect of cTnI-K118C mutation on early remodeling in cardiomyocytes.cardiac troponin I; culture of adult cardiomyocyte; prolongation of cardiomyocytes; myocardial remodeling TROPONIN I (TnI), the inhibitory subunit of the troponin complex, plays an essential role in the Ca 2ϩ regulation of striated muscle contraction (8,21,22). We previously reported a point mutation in cardiac TnI (cTnI) in wild turkeys that generates a single-amino acid substitution of Cys for Arg 111 (4). This residue is located in the ␣-helix interfacing with troponin T (TnT) in the I-T arm of the troponin complex (23, 26) and is highly conserved as Arg or Lys in the three muscle fiber type-specific (cardiac and slow and fast skeletal muscle) TnI isoforms in all vertebrate species examined (4, 12). Because of the variable length of the NH 2 -terminal segment, the residues in human and mouse cTnI corresponding to Arg 111 in turkey cTnI are Lys 117 and Lys 118 , respectively (12). Our previous studies demonstrated that the Arg 111 Cys substitution decreased binding affinity of turkey cTnI for cardiac TnT (4). Transgenic mouse...

show abstract

Section: Discussionmentioning

confidence: 89%

A dominantly negative mutation in cardiac troponin I at the interface with troponin T causes early remodeling in ventricular cardiomyocytes

Wei

Jin

2014

American Journal of Physiology-Cell Physiology

View full text Add to dashboard Cite

show abstract

“…Cardiomyocytes were isolated from adult mouse hearts as described previously (62). After isolation, cardiomyocytes were resuspended in plating medium (Table S1) and plated for 2 h as described below.…”

Section: Methodsmentioning

confidence: 99%

Dystrophin–glycoprotein complex regulates muscle nitric oxide production through mechanoregulation of AMPK signaling

Garbincius

Michele

2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Patients deficient in dystrophin, a protein that links the cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex (DGC), exhibit muscular dystrophy, cardiomyopathy, and impaired muscle nitric oxide (NO) production. We used live-cell NO imaging and in vitro cyclic stretch of isolated adult mouse cardiomyocytes as a model system to investigate if and how the DGC directly regulates the mechanical activation of muscle NO signaling. Acute activation of NO synthesis by mechanical stretch was impaired in dystrophin-deficient mdx cardiomyocytes, accompanied by loss of stretch-induced neuronal NO synthase (nNOS) S1412 phosphorylation. Intriguingly, stretch induced the acute activation of AMP-activated protein kinase (AMPK) in normal cardiomyocytes but not in mdx cardiomyocytes, and specific inhibition of AMPK was sufficient to attenuate mechanoactivation of NO production. Therefore, we tested whether direct pharmacologic activation of AMPK could bypass defective mechanical signaling to restore nNOS activity in dystrophin-deficient cardiomyocytes. Indeed, activation of AMPK with 5-aminoimidazole-4-carboxamide riboside or salicylate increased nNOS S1412 phosphorylation and was sufficient to enhance NO production in mdx cardiomyocytes. We conclude that the DGC promotes the mechanical activation of cardiac nNOS by acting as a mechanosensor to regulate AMPK activity, and that pharmacologic AMPK activation may be a suitable therapeutic strategy for restoring nNOS activity in dystrophin-deficient hearts and muscle.T he muscular dystrophies are a group of muscle wasting disorders characterized by progressive weakening and degeneration of striated muscle. The most common form is Duchenne muscular dystrophy (DMD), an X-linked disorder caused by genetic disruption of dystrophin (1) that affects 1 in 3,500-5,000 males (2, 3). DMD and several other types of muscular dystrophy result from disruption of the dystrophin-glycoprotein complex (DGC), a structure that spans the sarcolemma and forms a mechanical linkage between the cytoskeleton and the extracellular matrix via the association of dystrophin with subsarcolemmal γ-actin and the binding of α-dystroglycan to laminin (4). The generally accepted role for this complex is to act as a molecular shock absorber and stabilize the plasma membrane during muscle contraction. Disruption of the DGC's linkage between the cytoskeleton and extracellular matrix, such as occurs in DMD (1, 5-7) or with the disruption of α-dystroglycan-laminin binding in glycosylation-deficient muscular dystrophies (8, 9), leads to destabilization of the plasma membrane, rendering skeletal muscle fibers and cardiomyocytes susceptible to stretch-or contraction-induced injury and cell death (10)(11)(12)(13)(14).In addition to this structural role, a signaling function for the DGC has been proposed based on its association with several signaling proteins including Grb2-Sos1 (15), MEK and ERK (16), heterotrimeric G protein subunits (17, 18), archvillin (19), and neuronal nitric oxide synthase (n...

show abstract

“…We next examined whether PDE1C directly regulates cardiac myocyte hypertrophy. Isolated adult mouse cardiac myocytes from PDE1C-WT and -KO mice were stimulated with Ang II or ISO for 72 h in the presence of blebbistatin (a myosin II inhibitor) to block myocyte contraction and to extend their survival during culture (21). Myocyte hypertrophy was assessed by measuring the average cell area.…”

Section: Pde1c Expression In Failing Heartsmentioning

confidence: 99%

PDE1C deficiency antagonizes pathological cardiac remodeling and dysfunction

Knight

Chen

Zhang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Cyclic nucleotide phosphodiesterase 1C (PDE1C) represents a major phosphodiesterase activity in human myocardium, but its function in the heart remains unknown. Using genetic and pharmacological approaches, we studied the expression, regulation, function, and underlying mechanisms of PDE1C in the pathogenesis of cardiac remodeling and dysfunction. PDE1C expression is up-regulated in mouse and human failing hearts and is highly expressed in cardiac myocytes but not in fibroblasts. In adult mouse cardiac myocytes, PDE1C deficiency or inhibition attenuated myocyte death and apoptosis, which was largely dependent on cyclic AMP/PKA and PI3K/AKT signaling. PDE1C deficiency also attenuated cardiac myocyte hypertrophy in a PKA-dependent manner. Conditioned medium taken from PDE1C-deficient cardiac myocytes attenuated TGF-β-stimulated cardiac fibroblast activation through a mechanism involving the crosstalk between cardiac myocytes and fibroblasts. In vivo, cardiac remodeling and dysfunction induced by transverse aortic constriction, including myocardial hypertrophy, apoptosis, cardiac fibrosis, and loss of contractile function, were significantly attenuated in PDE1C-knockout mice relative to wild-type mice. These results indicate that PDE1C activation plays a causative role in pathological cardiac remodeling and dysfunction. Given the continued development of highly specific PDE1 inhibitors and the high expression level of PDE1C in the human heart, our findings could have considerable therapeutic significance.cyclic nucleotide | phosphodiesterase | cardiac remodeling | heart failure

show abstract

Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression

Abstract: Kabaeva Z, Zhao M, Michele DE. Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression.

Cited by 83 publications

References 35 publications

A dominantly negative mutation in cardiac troponin I at the interface with troponin T causes early remodeling in ventricular cardiomyocytes

A dominantly negative mutation in cardiac troponin I at the interface with troponin T causes early remodeling in ventricular cardiomyocytes

Dystrophin–glycoprotein complex regulates muscle nitric oxide production through mechanoregulation of AMPK signaling

PDE1C deficiency antagonizes pathological cardiac remodeling and dysfunction

Contact Info

Product

Resources

About