Background Heart failure (HF) is associated with excessive extracellular matrix (ECM) deposition and abnormal ECM degradation leading to cardiac fibrosis. Connective Tissue Growth Factor (CTGF) modulates ECM production during inflammatory tissue injury, but available data on CTGF gene expression in failing human heart and its response to mechanical unloading are limited. Methods and Results LV tissue from patients undergoing cardiac transplantation for ischemic (ICM; n=20) and dilated (DCM; n=20) cardiomyopathies, and from nonfailing (NF; n=20) donor hearts were examined. Paired samples (n=15) from patients undergoing LV assist device (LVAD) implantation as “bridge to transplant” (34-1145 days) were also analyzed. There was more interstitial fibrosis in both ICM and DCM compared to NF hearts. Hydroxyproline concentration was also significantly increased in DCM relative to NF samples. The expression of CTGF,TGFB1, COL1-A1, COL3-A1, MMP2 and MMP9 mRNAs in ICM and DCM were also significantly elevated as compared to NF controls. Although TGFB1, CTGF, COL1-A1, and COL3-A1 mRNA levels were reduced by unloading, there was only a modest reduction in tissue fibrosis and no difference in protein-bound hydroxyproline concentration between pre- and post-LVAD tissue samples. The persistent fibrosis may be related to a concomitant reduction in MMP9 mRNA and protein levels following unloading. Conclusions CTGF may be a key regulator of fibrosis during maladaptive remodeling and progression to HF. Although mechanical unloading normalizes most genotypic and functional abnormalities, its effect on ECM remodeling during HF is incomplete.

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marel. Isoenzyme-selective regulation of SERCA2 gene expression by protein kinase C in neonatal rat ventricular myocytes. Am J Physiol Cell Physiol 285: C39-C47, 2003. First published February 26, 2003 10.1152/ajpcell.00461. 2002-Patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA2) gene expression. We previously showed that SERCA2 downregulation can be simulated in cultured neonatal rat ventricular myocytes (NRVM) by treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). However, NRVM express three different PMA-sensitive PKC isoenzymes (PKC␣, PKC⑀, and PKC␦), which may be differentially regulated and have specific functions in the cardiomyocyte. Therefore, in this study we used adenoviral vectors encoding wild-type (wt) and kinase-defective, dominant negative (dn) mutant forms of PKC␣, PKC⑀, and PKC␦ to analyze their individual effects in regulating SERCA2 gene expression in NRVM. Overexpression of wtPKC⑀ and wtPKC␦, but not wtPKC␣, was sufficient to downregulate SERCA2 mRNA levels, as assessed by Northern blotting and quantitative, real-time RT-PCR (69 Ϯ 7 and 61 Ϯ 9% of control levels for wtPKC⑀ and wtPKC␦, respectively; P Ͻ 0.05 for each adenovirus; n ϭ 8 experiments). Conversely, overexpression of all three dnPKCs appeared to significantly increase SERCA2 mRNA levels (dnPKC␦ Ͼ dnPKC⑀ Ͼ dnPKC␣). dnPKC␦ overexpression produced the largest increase (2.8 Ϯ 1.0-fold; n ϭ 11 experiments). However, PMA treatment was still sufficient to downregulate SERCA2 mRNA levels despite overexpression of each dominant negative mutant. These data indicate that the novel PKC isoenzymes PKC⑀ and PKC␦ selectively regulate SERCA2 gene expression in cardiomyocytes but that neither PKC alone is necessary for this effect if the other novel PKC can be activated.heart; signal transduction; hypertrophy; transcription; mRNA stability; sarco(endo)plasmic reticulum Ca 2ϩ -ATPase HEART FAILURE (HF) is the final common syndrome of most primary cardiovascular diseases, including coronary atherosclerosis, hypertension, cardiomyopathy, and valvular and congenital heart malformations (17). Despite the varied primary causes of HF, mechanical overload, increased wall stress, and neurohormonal activation are common features that precede the HF state. In response to these mechanical and neurohormonal stimuli, the process of ventricular remodeling is initiated, which is associated with numerous changes in myocardial gene expression and protein turnover. Cultured neonatal rat ventricular myocytes (NRVM) have proven to be useful tools in understanding the cellular mechanisms regulating SERCA2 gene expression in response to neurohormonal and mechanical stimuli. For instance, previous studies have shown that SERCA2 gene expression is regulated by peptide growth factors (33), thyroid hormones (24), angiotensin II (28), endothelin-1 (ET) (23), and norepinephrine (9). Studies fro...

Proline-rich tyrosine kinase 2 (PYK2) is a member of the focal adhesion kinase (FAK) family of nonreceptor protein tyrosine kinases. PYK2 has been implicated in linking G protein-coupled receptors to activation of mitogen-activated protein kinase cascades and cellular growth in a variety of cell types. To determine whether PYK2 expression and phosphorylation is altered in left ventricular (LV) myocardium undergoing LV hypertrophy (LVH) and heart failure in vivo, suprarenal abdominal aortic coarctation was performed in 160-g male Sprague-Dawley rats. Immunohistochemistry and Western blotting were performed on LV tissue 1, 8, and 24 wk after aortic banding. Aortic banding produced sustained hypertension and gradually developing LVH. PYK2 levels were increased 1.8 Ϯ 0.2-, 2.7 Ϯ 0.6-, and 2.0 Ϯ 0.2-fold in 1-, 8-, and 24-wk banded animals compared with their respective sham-operated controls. The increase in PYK2 expression was paralleled by an increase in PYK2 phosphorylation, both of which preceded the development of LVH. Immunohistochemistry revealed that enhanced PYK2 expression occurred predominantly in the cardiomyocyte population. Furthermore, there was a high degree of correlation (R ϭ 0.75; P Ͻ 0.001) between the level of PYK2 and the degree of LVH in 24-wk sham and banded animals. In contrast, FAK levels and FAK phosphorylation were not increased before the development of LVH. However, there was a high degree of correlation (R ϭ 0.68; P Ͻ 0.001) between the level of FAK and the degree of LVH in 24-wk sham and banded rats. There was also a significant increase in the ratio of phosphospecific anti-FAK to FAK at this time point. These data are consistent with a role for PYK2 in the induction of pressure overload-induced cardiomyocyte hypertrophy, and suggest that PYK2 and FAK have distinctly different roles in LVH progression. signal transduction; cytoskeleton; heart failure; focal adhesion kinase CARDIAC HYPERTROPHY is a compensatory process that leads to a heart better suited for the functional demands caused by myocyte dysfunction or loss. Because cardiac myocytes are terminally differentiated and nonproliferative, hypertrophic myocytes demonstrate an increase in protein content rather than an increase in myocyte number. Cardiac hypertrophy is also associated with sarcomeric reorganization and characteristic changes in gene expression and protein turnover. Cardiac hypertrophy, however, may independently lead to cardiac dysfunction and is also a common feature associated with the heart failure state. Despite advancements in the treatment and understanding of the pathogenesis of heart failure, the cellular and molecular mechanisms involved in hypertrophy induction are still largely unknown.Recent studies (12,17,18) have suggested that nonreceptor protein tyrosine kinases are important in transducing mechanical stimuli to biochemical pathways that lead to cardiomyocyte hypertrophy. Prolinerich tyrosine kinase 2 (PYK2) and focal adhesion kinase (FAK) are members of the FAK family of nonreceptor protein tyrosin...

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Cardiomyocytes express several isoenzymes of protein kinase C (PKC), which as a group have been implicated in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. Individual PKC isoenzymes also require transphosphorylation and autophosphorylation for enzymatic activity. To determine whether PKC isoenzyme expression and autophosphorylation are altered during LVH progression in vivo, suprarenal abdominal aortic coarctation was performed in Sprague-Dawley rats. Quantitative Western blotting was performed on LV tissue 1, 8 and 24 weeks after aortic banding, using antibodies specific for total PKCalpha, PKCdelta and PKCepsilon, and their C-terminal autophosphorylation sites. Aortic banding produced sustained hypertension and gradually developing LVH that progressed to diastolic heart failure over time. PKCepsilon levels and autophosphorylation were not significantly different from sham-operated controls during any stage of LVH progression. PKCalpha expression levels were also unaffected during the induction of LVH, but increased 3.2 +/- 0.8 fold during the transition to heart failure. In addition, there was a high degree of correlation between PKCalpha levels and the degree of LVH in 24 week banded animals. However, autophosphorylated PKCalpha was not increased at any time point. In contrast, PKCdelta autophosphorylation was increased prior to the development of LVH, and also during the transition to heart failure. The increased PKCdelta autophosphorylation in 1 week banded rats was not accompanied by an increase in total PKCdelta, whereas total PKCdelta levels were markedly increased (6.0 +/- 1.7 fold) in 24 week banded animals. Furthermore, both phosphorylated and total PKCdelta levels were highly correlated with the degree of LVH in 24 week banded rats. In summary, we provide indirect evidence to indicate that PKCdelta may be involved in the induction of pressure overload LVH, whereas both PKCdelta and PKCalpha may be involved in the transition to heart failure.

Koczor CA, Torres RA, Fields EJ, Boyd A, He S, Patel N, Lee EK, Samarel AM, Lewis W. Thymidine kinase and mtDNA depletion in human cardiomyopathy: epigenetic and translational evidence for energy starvation. Physiol Genomics 45: 590 -596, 2013. First published May 21, 2013 doi:10.1152/physiolgenomics.00014.2013.-This study addresses how depletion of human cardiac left ventricle (LV) mitochondrial DNA (mtDNA) and epigenetic nuclear DNA methylation promote cardiac dysfunction in human dilated cardiomyopathy (DCM) through regulation of pyrimidine nucleotide kinases. Samples of DCM LV and right ventricle (n ϭ 18) were obtained fresh at heart transplant surgery. Parallel samples from nonfailing (NF) controls (n ϭ 12) were from donor hearts found unsuitable for clinical use. We analyzed abundance of mtDNA and nuclear DNA (nDNA) using qPCR. LV mtDNA was depleted in DCM (50%, P Ͻ 0.05 each) compared with NF. No detectable change in RV mtDNA abundance occurred. DNA methylation and gene expression were determined using microarray analysis (GEO accession number: GSE43435). Fifty-seven gene promoters exhibited DNA hypermethylation or hypomethylation in DCM LVs. Among those, cytosolic thymidine kinase 1 (TK1) was hypermethylated. Expression arrays revealed decreased abundance of the TK1 mRNA transcript with no change in transcripts for other relevant thymidine metabolism enzymes. Quantitative immunoblots confirmed decreased TK1 polypeptide steady state abundance. TK1 activity remained unchanged in DCM samples while mitochondrial thymidine kinase (TK2) activity was significantly reduced. Compensatory TK activity was found in cardiac myocytes in the DCM LV. Diminished TK2 activity is mechanistically important to reduced mtDNA abundance and identified in DCM LV samples here. Epigenetic and genetic changes result in changes in mtDNA and in nucleotide substrates for mtDNA replication and underpin energy starvation in DCM.