Serum circulating proteins from pediatric patients with dilated cardiomyopathy cause pathologic remodeling and cardiomyocyte stiffness
Danielle A. Jeffrey, Julie Pires Da Silva, Anastacia M. Garcia, Xuan Jiang, Anis Karimpour-Fard, Lee S. Toni, Thomas Lanzicher, Brisa Peña, Carissa A. Miyano, Karin Nunley, Armin Korst, Orfeo Sbaizero, Matthew R.G. Taylor, Shelley D. Miyamoto, Brian L. Stauffer, Carmen C. Sucharov
Danielle A. Jeffrey, Julie Pires Da Silva, Anastacia M. Garcia, Xuan Jiang, Anis Karimpour-Fard, Lee S. Toni, Thomas Lanzicher, Brisa Peña, Carissa A. Miyano, Karin Nunley, Armin Korst, Orfeo Sbaizero, Matthew R.G. Taylor, Shelley D. Miyamoto, Brian L. Stauffer, Carmen C. Sucharov
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Research Article Cardiology

Serum circulating proteins from pediatric patients with dilated cardiomyopathy cause pathologic remodeling and cardiomyocyte stiffness

Abstract

Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy and main indication for heart transplantation in children. Therapies specific to pediatric DCM remain limited due to lack of a disease model. Our previous study showed that treatment of neonatal rat ventricular myocytes (NRVMs) with serum from nonfailing or DCM pediatric patients activates the fetal gene program (FGP). Here we show that serum treatment with proteinase K prevents activation of the FGP, whereas RNase treatment exacerbates it, suggesting that circulating proteins, but not circulating miRNAs, promote these pathological changes. Evaluation of the protein secretome showed that midkine (MDK) is upregulated in DCM serum, and NRVM treatment with MDK activates the FGP. Changes in gene expression in serum-treated NRVMs, evaluated by next-generation RNA-Seq, indicated extracellular matrix remodeling and focal adhesion pathways were upregulated in pediatric DCM serum and in DCM serum–treated NRVMs, suggesting alterations in cellular stiffness. Cellular stiffness was evaluated by Atomic Force Microscopy, which showed an increase in stiffness in DCM serum–treated NRVMs. Of the proteins increased in DCM sera, secreted frizzled-related protein 1 (sFRP1) was a potential candidate for the increase in cellular stiffness, and sFRP1 treatment of NRVMs recapitulated the increase in cellular stiffness observed in response to DCM serum treatment. Our results show that serum circulating proteins promoted pathological changes in gene expression and cellular stiffness, and circulating miRNAs were protective against pathological changes.

Authors

Danielle A. Jeffrey, Julie Pires Da Silva, Anastacia M. Garcia, Xuan Jiang, Anis Karimpour-Fard, Lee S. Toni, Thomas Lanzicher, Brisa Peña, Carissa A. Miyano, Karin Nunley, Armin Korst, Orfeo Sbaizero, Matthew R.G. Taylor, Shelley D. Miyamoto, Brian L. Stauffer, Carmen C. Sucharov

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Figure 2

Fetal gene program expression changes in DCM serum– and NF serum–treated NRVMs with or without DNase and RNase.

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Fetal gene program expression changes in DCM serum– and NF serum–treated...
(A) RT-qPCR of FGP expression in NRVMs transfected with serum with or without DNase. Expression of BNP, ANF, and α-MyHC to β-MyHC ratios. Gene expression was normalized to 18S, and data are presented as a relative fold change to NF controls. DCM n = 8, NF n = 4 from 7 independent NRVM preps. (B) RT-qPCR of FGP expression analysis of serum-treated NVRMs with or without RNase. Expression of BNP, ANF, and α-MyHC/β-MyHC was evaluated. Gene expression was normalized to 18S, and data are presented as a relative fold change to NF controls. DCM n = 9, NF n = 5 from 9 independent NRVM preps. All groups are log2 transformed and error bar denotes mean ± SEM. P values are notated in the figure. Fitting a mixed model, Sidak’s multiple comparisons test was used for all data sets. DCM, dilated cardiomyopathy; NRVMs, neonatal rat ventricular myocytes; NF, nonfailing; FGP, fetal gene program; BNP, natriuretic peptide B; ANF, atrial natriuretic factor; α-MyHC, α-myosin heavy chain; β-MyHC, β-myosin heavy chain.