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

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.

A PDE3A Promoter Polymorphism Regulates cAMP-Induced Transcriptional Activity in Failing Human Myocardium.

BACKGROUND: The phosphodiesterase 3A (PDE3A) gene encodes a PDE that regulates cardiac myocyte cyclic adenosine monophosphate (cAMP) levels and myocardial contractile function. PDE3 inhibitors (PDE3i) are used for short-term treatment of refractory heart failure (HF), but do not produce uniform long-term benefit. OBJECTIVES: The authors tested the hypothesis that drug target genetic variation could explain clinical response heterogeneity to PDE3i in HF. METHODS: PDE3A promoter studies were performed in a cloned luciferase construct. In human left ventricular (LV) preparations, mRNA expression was measured by reverse transcription polymerase chain reaction, and PDE3 enzyme activity by cAMP-hydrolysis. RESULTS: The authors identified a 29-nucleotide (nt) insertion (INS)/deletion (DEL) polymorphism in the human PDE3A gene promoter beginning 2,214 nt upstream from the PDE3A1 translation start site. Transcription factor ATF3 binds to the INS and represses cAMP-dependent promoter activity. In explanted failing LVs that were homozygous for PDE3A DEL and had been treated with PDE3i pre-cardiac transplantation, PDE3A1 mRNA abundance and microsomal PDE3 enzyme activity were increased by 1.7-fold to 1.8-fold (p < 0.05) compared with DEL homozygotes not receiving PDE3i. The basis for the selective up-regulation in PDE3A gene expression in DEL homozygotes treated with PDE3i was a cAMP response element enhancer 61 nt downstream from the INS, which was repressed by INS. The DEL homozygous genotype frequency was also enriched in patients with HF. CONCLUSIONS: A 29-nt INS/DEL polymorphism in the PDE3A promoter regulates cAMP-induced PDE3A gene expression in patients treated with PDE3i. This molecular mechanism may explain response heterogeneity to this drug class, and may inform a pharmacogenetic strategy for a more effective use of PDE3i in HF.

Phosphodiesterase-5 Is Elevated in Failing Single Ventricle Myocardium and Affects Cardiomyocyte Remodeling In Vitro.

Background Single ventricle (SV) congenital heart disease is fatal without intervention, and eventual heart failure is a major cause of morbidity and mortality. Although there are no proven medical therapies for the treatment or prevention of heart failure in the SV heart disease population, phosphodiesterase-5 inhibitors (PDE5i), such as sildenafil, are increasingly used. Although the pulmonary vasculature is the primary target of PDE5i therapy in patients with SV heart disease, the effects of PDE5i on the SV heart disease myocardium remain largely unknown. We sought to determine PDE5 expression and activity in the single right ventricle of SV heart disease patients relative to nonfailing controls and to determine whether PDE5 impacts cardiomyocyte remodeling using a novel serum-based in vitro model. Methods and Results PDE5 expression (n=9 nonfailing; n=7 SV heart disease), activity (n=8 nonfailing; n=9 SV heart disease), and localization (n=3 SV heart disease) were determined in explanted human right ventricle myocardium. PDE5 is expressed in SV heart disease cardiomyocytes, and PDE5 protein expression and activity are increased in SV heart disease right ventricle compared with nonfailing right ventricle. Isolated neonatal rat ventricular myocytes were treated for 72 hours with nonfailing or SV heart disease patient serum±sildenafil. Reverse transcription quantitative polymerase chain reaction (n=5 nonfailing; n=12 SV heart disease) and RNA sequencing (n=3 nonfailing; n=3 SV heart disease) were performed on serum-treated neonatal rat ventricular myocytes and demonstrated that treatment with SV heart disease sera results in pathological gene expression changes that are attenuated with PDE5i. Conclusions PDE5 is increased in failing SV heart disease myocardium, and pathological gene expression changes in SV heart disease serum-treated neonatal rat ventricular myocytes are abrogated by PDE5i. These results suggest that PDE5 represents an intriguing myocardial therapeutic target in this population.

Fibrosis-Related Gene Expression in Single Ventricle Heart Disease.

OBJECTIVE: To evaluate fibrosis and fibrosis-related gene expression in the myocardium of pediatric subjects with single ventricle with right ventricular failure. STUDY DESIGN: Real-time quantitative polymerase chain reaction was performed on explanted right ventricular myocardium of pediatric subjects with single ventricle disease and controls with nonfailing heart disease. Subjects were divided into 3 groups: single ventricle failing (right ventricular failure before or after stage I palliation), single ventricle nonfailing (infants listed for primary transplantation with normal right ventricular function), and stage III (Fontan or right ventricular failure after stage III). To evaluate subjects of similar age and right ventricular volume loading, single ventricle disease with failure was compared with single ventricle without failure and stage III was compared with nonfailing right ventricular disease. Histologic fibrosis was assessed in all hearts. Mann-Whitney tests were performed to identify differences in gene expression. RESULTS: Collagen (Col1α, Col3) expression is decreased in single ventricle congenital heart disease with failure compared with nonfailing single ventricle congenital heart disease (P = .019 and P = .035, respectively), and is equivalent in stage III compared with nonfailing right ventricular heart disease. Tissue inhibitors of metalloproteinase (TIMP-1, TIMP-3, and TIMP-4) are downregulated in stage III compared with nonfailing right ventricular heart disease (P = .0047, P = .013 and P = .013, respectively). Matrix metalloproteinases (MMP-2, MMP-9) are similar between nonfailing single ventricular heart disease and failing single ventricular heart disease, and between stage III heart disease and nonfailing right ventricular heart disease. There is no difference in the prevalence of right ventricular fibrosis by histology in subjects with single ventricular failure heart disease with right ventricular failure (18%) compared with those with normal right ventricular function (38%). CONCLUSIONS: Fibrosis is not a primary contributor to right ventricular failure in infants and young children with single ventricular heart disease. Additional studies are required to understand whether antifibrotic therapies are beneficial in this population.

Pediatric dilated cardiomyopathy hearts display a unique gene expression profile.

Our previous work showed myocellular differences in pediatric and adult dilated cardiomyopathy (DCM). However, a thorough characterization of the molecular pathways involved in pediatric DCM does not exist, limiting the development of age-specific therapies. To characterize this patient population, we investigated the transcriptome profile of pediatric patients. RNA-Seq from 7 DCM and 7 nonfailing (NF) explanted age-matched pediatric left ventricles (LV) was performed. Changes in gene expression were confirmed by real-time PCR (RT-PCR) in 36 DCM and 21 NF pediatric hearts and in 20 DCM and 10 NF adult hearts. The degree of myocyte hypertrophy was investigated in 4 DCM and 7 NF pediatric hearts and in 4 DCM and 9 NF adult hearts. Changes in gene expression in response to pluripotency-inducing factors were investigated in neonatal rat ventricular myocytes (NRVMs). Transcriptome analysis identified a gene expression profile in children compared with adults with DCM. Additionally, myocyte hypertrophy was not observed in pediatric hearts but was present in adult hearts. Furthermore, treatment of NRVMs with pluripotency-inducing factors recapitulated changes in gene expression observed in the pediatric DCM heart. Pediatric DCM is characterized by unique changes in gene expression that suggest maintenance of an undifferentiated state.

Histone deacetylase adaptation in single ventricle heart disease and a young animal model of right ventricular hypertrophy.

BackgroundHistone deacetylase (HDAC) inhibitors are promising therapeutics for various forms of cardiac diseases. The purpose of this study was to assess cardiac HDAC catalytic activity and expression in children with single ventricle (SV) heart disease of right ventricular morphology, as well as in a rodent model of right ventricular hypertrophy (RVH).MethodsHomogenates of right ventricle (RV) explants from non-failing controls and children born with a SV were assayed for HDAC catalytic activity and HDAC isoform expression. Postnatal 1-day-old rat pups were placed in hypoxic conditions, and echocardiographic analysis, gene expression, HDAC catalytic activity, and isoform expression studies of the RV were performed.ResultsClass I, IIa, and IIb HDAC catalytic activity and protein expression were elevated in the hearts of children born with a SV. Hypoxic neonatal rats demonstrated RVH, abnormal gene expression, elevated class I and class IIb HDAC catalytic activity, and protein expression in the RV compared with those in the control.ConclusionsThese data suggest that myocardial HDAC adaptations occur in the SV heart and could represent a novel therapeutic target. Although further characterization of the hypoxic neonatal rat is needed, this animal model may be suitable for preclinical investigations of pediatric RV disease and could serve as a useful model for future mechanistic studies.

Myocardial microRNAs associated with reverse remodeling in human heart failure.

BACKGROUND: In dilated cardiomyopathies (DCMs) changes in expression of protein-coding genes are associated with reverse remodeling, and these changes can be regulated by microRNAs (miRs). We tested the general hypothesis that dynamic changes in myocardial miR expression are predictive of ß-blocker-associated reverse remodeling. METHODS: Forty-three idiopathic DCM patients (mean left ventricular ejection fraction 0.24 ± 0.09) were treated with ß-blockers. Serial ventriculography and endomyocardial biopsies were performed at baseline, and after 3 and 12 months of treatment. Changes in RT-PCR (candidate miRs) or array-measured miRs were compared based on the presence (R) or absence (NR) of a reverse-remodeling response, and a miR-mRNA-function pathway analysis (PA) was performed. RESULTS: At 3 months, 2 candidate miRs were selectively changed in Rs, decreases in miR-208a-3p and miR-591. PA revealed changes in miR-mRNA interactions predictive of decreased apoptosis and myocardial cell death. At 12 months, 5 miRs exhibited selective changes in Rs (decreases in miR-208a-3p, -208b-3p, 21-5p, and 199a-5p; increase in miR-1-3p). PA predicted decreases in apoptosis, cardiac myocyte cell death, hypertrophy, and heart failure, with increases in contractile and overall cardiac functions. CONCLUSIONS: In DCMs, myocardial miRs predict the time-dependent reverse-remodeling response to ß-blocker treatment, and likely regulate the expression of remodeling-associated miRs. TRIAL REGISTRATION: ClinicalTrials.gov NCT01798992. FUNDING: NIH 2R01 HL48013, 1R01 HL71118 (Bristow, PI); sponsored research agreements from Glaxo-SmithKline and AstraZeneca (Bristow, PI); NIH P20 HL101435 (Lowes, Port multi-PD/PI); sponsored research agreement from Miragen Therapeutics (Port, PI).

Cardiac Adenylyl Cyclase and Phosphodiesterase Expression Profiles Vary by Age, Disease, and Chronic Phosphodiesterase Inhibitor Treatment.

BACKGROUND: Pediatric heart failure (HF) patients have a suboptimal response to traditional HF medications, although phosphodiesterase-3 inhibition (PDE3i) has been used with greater success than in the adult HF population. We hypothesized that molecular alterations specific to children with HF and HF etiology may affect response to treatment. METHODS AND RESULTS: Adenylyl cyclase (AC) and phosphodiesterase (PDE) isoforms were quantified by means of quantitative real-time polymerase chain reaction in explanted myocardium from adults with dilated cardiomyopathy (DCM), children with DCM, and children with single-ventricle congenital heart disease of right ventricular morphology (SRV). AC and PDE expression profiles were uniquely regulated in each subject group and demonstratde distinct changes in response to chronic PDE3i. There was unique up-regulation of AC5 in adult DCM with PDE3i (fold change 2.415; P = .043), AC2 in pediatric DCM (fold change 2.396; P = .0067), and PDE1C in pediatric SRV (fold change 1.836; P = .032). Remarkably, PDE5A expression was consistently increased across all age and disease groups. CONCLUSIONS: Unique regulation of AC and PDE isoforms supports a differential molecular adaptation to HF in children compared with adults, and may help identify mechanisms specific to the pathogenesis of pediatric HF. Greater understanding of these differences will help optimize medical therapies based on age and disease process.

Micro-RNA expression in hypoplastic left heart syndrome.

OBJECTIVE: Micro-RNAs (miRNAs) are important regulators of gene expression through interaction with the 3'UTR of target messenger RNAs (mRNAs). The role of miRNAs has been extensively studied in adult human and nonhuman animal models of heart disease. Hypoplastic left heart syndrome (HLHS) is the most common form of severe congenital heart disease and is an important cause of morbidity and mortality in infants and children. The objective of this work was to analyze the miRNA profile in HLHS patients. METHODS AND RESULTS: miRNA profile was determined in the right ventricle with the use of miRNA array, and expression was validated with the use of reverse-transcription polymerase chain reaction (RT-PCR). Based on bioinformatics analysis, targets were selected and their expression analyzed with the use of RT-PCR.We found that the miRNA profile of HLHS is novel, with few similarities between pediatric and adult idiopathic dilated cardiomyopathy. Moreover, our analysis identified putative targets for these miRNAs that are known to be important for cardiac development and disease, and that miRNAs and their putative targets are antithetically regulated. We also found that miRNA expression changes with stage of surgery, suggesting that volume unloading of the ventricle has important consequences for gene expression. CONCLUSIONS: Our data suggest a unique miRNA profile for HLHS that may be associated with defects in cardiac development and disease.

Gene expression and ß-adrenergic signaling are altered in hypoplastic left heart syndrome.

BACKGROUND: The purpose of the current study was to define the myocellular changes and adaptation of the ß-adrenergic receptor (ß-AR) system that occur in the systemic right ventricle (RV) of children with hypoplastic left heart syndrome (HLHS). METHODS: Explanted hearts from children with HLHS and non-failing controls were used for this study. HLHS patients were divided into 2 groups: "compensated" (C-HLHS), infants listed for primary transplant with normal RV function and absence of heart failure symptoms, and "decompensated" (D-HLHS), patients listed for transplant after failed surgical palliation with RV failure and/or refractory protein-losing enteropathy or plastic bronchitis. RESULTS: Compared with non-failing control RVs, the HLHS RV demonstrated decreased sarcoplasmic reticulum calcium-adenosine triphosphatase 2a and α-myosin heavy chain (MHC) gene expression, decreased total ß-AR due to down-regulation of ß1-AR, preserved cyclic adenosine monophosphate levels, and increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity. There was increased atrial natriuretic peptide expression only in the C-HLHS group. Unique to those in the D-HLHS group was increased ß-MHC and decreased α-MHC protein expression (MHC isoform switching), increased adenylyl cyclase 5 expression, and increased phosphorylation of the CaMK target site on phospholamban, threonine 17. CONCLUSIONS: The HLHS RV has an abnormal myocardial gene expression pattern, downregulation of ß1-AR, preserved cyclic adenosine monophosphate levels, and increased CaMKII activity compared with the non-failing control RV. There is MHC isoform switching, increased adenylyl cyclase 5, and increased phosphorylation of phospholamban threonine 17 only in the D-HLHS group. Although abnormal gene expression and changes in the ß-AR system precede clinically evident ventricular failure in HLHS, additional unique adaptations occur in those with HLHS and failed surgical palliation.

Beta-adrenergic adaptation in paediatric idiopathic dilated cardiomyopathy.

BACKGROUND: Although the pathophysiology and treatment of adult heart failure (HF) are well studied, HF in children remains poorly understood. In adults, adrenergic receptor (AR)-mediated adaptation plays a central role in cardiac abnormalities in HF, and these patients respond well to ß-blocker (BB) therapy. However, in children with HF, there is a growing body of literature suggesting a lack of efficacy of adult HF therapies. Due to these unanticipated differences in response to therapy and the paucity of data regarding the molecular adaptation of the paediatric heart, we investigated the molecular characteristics of HF in children. METHODS AND RESULTS: Explanted hearts from adults and children with idiopathic dilated cardiomyopathy and non-failing controls were used in the study. Our results show that the molecular characteristics of paediatric HF are strikingly different from their adult counterparts. These differences include: (i) down-regulation of ß1- and ß2-AR in children, whereas ß2-AR expression is maintained in adults; (ii) up-regulation of connexin43 in children, whereas down-regulation is observed in adults; (iii) no differences in phosphatase expression, whereas up-regulation is observed in adults; (iv) no decrease in the phosphorylation of phospholamban at the Ser16 or Thr17 sites in children, which are known characteristics of adult HF. CONCLUSION: There is a different adaptation of ß-AR and adrenergic signalling pathways in children with HF compared with adults. Our results begin to address the disparities in cardiovascular research specific to children and suggest that age-related differences in adaptation could influence the response to therapy. These findings could lead to a paradigm shift in the contemporary management of children with HF.

miRNA expression in pediatric failing human heart.

miRNAs are short regulatory RNAs that can regulate gene expression through interacting with the 3'UTR of target mRNAs. Although the role of miRNAs has been extensively studied in adult human and animal models of heart disease, nothing is known about their expression in pediatric heart failure patients. Different than adults with heart failure, pediatric patients respond well to phosphodiesterase inhibitor (PDEi) treatment, which is safe in the outpatient setting, results in fewer heart failure emergency department visits, fewer cardiac hospital admissions and improved NYHA classification. We have recently shown that pediatric heart failure patients display a unique molecular profile that is different from adults with heart failure. In this study we show for the first time that pediatric heart failure patients display a unique miRNA profile, and that expression of some miRNAs correlate with response to PDEi treatment. Moreover, we show that expression of Smad4, a potential target for PDEi-regulated miRNAs, is normalized in PDEi-treated patients. Since miRNAs may be used as therapy for human heart failure, our results underscore the importance of defining the molecular characteristics of pediatric heart failure patients, so age-appropriate therapy can be designed for this population.

Temporal analysis of mRNA and miRNA expression in transgenic mice overexpressing Arg- and Gly389 polymorphic variants of the ß1-adrenergic receptor.

Several studies in humans or transgenic animals have reported that the 389 Arg or Gly polymorphic variation of the ß1-adrenergic receptor (AR) is associated with differential responses to beta-blocker therapy and/or myocardial disease progression. Analysis of changes in gene expression is an important means of defining molecular differences associated with structural or functional phenotypic variations. To determine if structural and functional myocardial phenotypic differences between ß1389 Arg vs. Gly transgenic overexpressors are associated with qualitative and/or quantitative differences in gene expression, a comprehensive analysis of mRNAs and miRNAs expressed in the hearts of 3 and 6-8 mo old ß1-Arg389 and ß1-Gly389 overexpressor transgenic mice was performed. Changes in mRNA and miRNA expression were analyzed by arrays and partially confirmed by RT-qPCR. Bioinformatic analysis demonstrated that several genes, including those involved in PKA and CaMK signaling pathways, are regulated in a temporal- or phenotype-specific manner. Furthermore, expression signature analyses indicated that miRNAs have the potential to target expression of a number of genes involved in multiple cardiomyopathy-related pathways, and changes in miRNA expression can precede the onset of disease. Differences in gene expression between ß1-Arg389 and ß1-Gly389 transgenic mice are largely quantitative rather than qualitative and are associated with the development of cardiomyopathy in a time-dependent manner. Chronic ß1-AR overdrive results in increased expression of components of the CaMK pathway, with correspondingly decreased levels of components of the PKA pathway. Based on the temporal and genotype-specific pattern of miRNA expression, miRNAs are likely to be important predictors of disease states, especially when miRNA expression is paired with mRNA expression, and that miRNA/mRNA expression signatures have the potential to be useful in determining the underlying risk associated with cardiac disease progression.

Temporal expression of miRNAs and mRNAs in a mouse model of myocardial infarction.

Analysis of changes in gene expression is an important means to define molecular differences associated with the phenotypic changes observed in response to myocardial infarction (MI). Several studies in humans or animal models have reported differential miRNA expression in response to MI acutely (animal) or chronically (human). To determine the relative contribution of microRNA (miRNA) and mRNAs to acute and chronic temporal changes in response to MI, mRNA and miRNA expression profiles were performed in three time points post-MI. Changes in mRNA and miRNA expression was analyzed by arrays and confirmed by RT-PCR. Bioinformatic analysis demonstrated that several genes and miRNAs in various pathways are regulated in a temporal or phenotype-specific manner. Furthermore miRNA analyses indicated that miRNAs can target expression of several genes involved in multiple cardiomyopathy-related pathways. Our results suggest that: 1) Differentially regulated miRNAs are predicted to target expression of several genes in multiple biological processes involved in the response to MI; 2) antithetical and compensatory changes in miRNA expression are observed at later disease stages, including antithetical regulation of miR-29, which correlates with the expression of collagen genes, and upregulation of apoptosis-related miRNAs at early stages and antiapoptotic/growth promoting miRNAs at later stages; 3) temporally dependent changes in miRNA and mRNA expression post-MI are generally characterized by dramatic changes acutely postinjury and are normalized as disease progresses; 4) A combinatorial analysis of mRNA and miRNA expression may aid in determining factors involved in compensatory and decompensated responses to cardiac injury.

ß-Adrenergic receptor stimulation and activation of protein kinase A protect against α1-adrenergic-mediated phosphorylation of protein kinase D and histone deacetylase 5.

INTRODUCTION: Chronic activation of ß(1)-adrenergic receptor (ß(1)-AR) signaling can have deleterious effects on the heart, and animal models overexpressing ß(1)-ARs develop a dilated cardiomyopathy and heart failure. In the classic ß-AR pathway, receptor occupancy by an agonist results in increased cyclic adenosine monophosphate (cAMP) levels and activation of protein kinase A (PKA). However, the role of PKA-dependent signaling in the development and progression of cardiomyopathies and heart failure is controversial, because ß-AR signal transduction is generally desensitized in the failing heart and PKA activity is not increased. METHODS AND RESULTS: Neonatal rat ventricular myocytes were acutely (15 minutes) or chronically (48 hours) treated with isoproterenol, and phosphorylation of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) was measured. Acute ß(1)-AR stimulation or expression of constitutively active (CA) PKA reduced α(1)-adrenergic-mediated phosphorylation of HDAC5 and PKD by activation of a phosphatase. Overexpression of CA-PKA also reduced α(1)-adrenergic-mediated increased expression of contractile protein fetal isoforms and promoted repression of adult isoforms, but had no effect on α(1)-adrenergic-mediated cellular hypertrophy. CONCLUSIONS: These data indicate that the PKA-dependent arm of ß-AR signaling can be antihypertrophic and presumably beneficial, through dephosphorylation of PKD and HDAC5 and reduction of hypertrophic fetal isoform gene expression.

A beta1-adrenergic receptor CaM kinase II-dependent pathway mediates cardiac myocyte fetal gene induction.

Beta-adrenergic signaling plays an important role in the natural history of dilated cardiomyopathies. Chronic activation of beta-adrenergic receptors (beta1-AR and beta2-AR) during periods of cardiac stress ultimately harms the failing heart by mechanisms that include alterations in gene expression. Here, we show that stimulation of beta-ARs with isoproterenol in neonate rat ventricular myocytes causes a "fetal" response in the relative activities of the human cardiac fetal and/or adult gene promoters that includes repression of the human and rat alpha-myosin heavy chain (alpha-MyHC) promoters with simultaneous activation of the human atrial natriuretic peptide (ANP) and rat beta-MyHC promoters. We also show that the promoter changes correlate with changes in endogenous gene expression as measured by mRNA expression. Furthermore, we show that these changes are specifically mediated by the beta1-AR, but not the beta2-AR, and are independent of alpha1-AR stimulation. We also demonstrate that the fetal gene response is independent of cAMP and protein kinase A, whereas inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK) pathway blocks isoproterenol-mediated fetal gene program induction. Finally, we show that induction of the fetal program is dependent on activation of the L-type Ca2+ channel. We conclude that in neonatal rat cardiac myocytes, agonist-occupied beta1-AR mobilizes Ca2+ stores to activate fetal gene induction through cAMP independent pathways that involve CaMK.

The PPAR-alpha activator fenofibrate fails to provide myocardial protection in ischemia and reperfusion in pigs.

Rodent studies suggest that peroxisome proliferator-activated receptor-alpha (PPAR-alpha) activation reduces myocardial ischemia-reperfusion (I/R) injury and infarct size; however, effects of PPAR-alpha activation in large animal models of myocardial I/R are unknown. We determined whether chronic treatment with the PPAR-alpha activator fenofibrate affects myocardial I/R injury in pigs. Domestic farm pigs were assigned to treatment with fenofibrate 50 mg.kg(-1).day(-1) orally or no drug treatment, and either a low-fat (4% by weight) or a high-fat (20% by weight) diet. After 4 wk, 66 pigs underwent 90 min low-flow regional myocardial ischemia and 120 min reperfusion under anesthetized open-chest conditions, resulting in myocardial stunning. The high-fat group received an infusion of triglyceride emulsion and heparin during this terminal experiment to maintain elevated arterial free fatty acid (FFA) levels. An additional 21 pigs underwent 60 min no-flow ischemia and 180 min reperfusion, resulting in myocardial infarction. Plasma concentration of fenofibric acid was similar to the EC50 for activation of PPAR-alpha in vitro and to maximal concentrations achieved in clinical use. Myocardial expression of PPAR-alpha mRNA was prominent but unaffected by fenofibrate treatment. Fenofibrate increased expression of carnitine palmitoyltransferase (CPT)-I mRNA in liver and decreased arterial FFA and lactate concentrations (each P < 0.01). However, fenofibrate did not affect myocardial CPT-I expression, substrate uptake, lipid accumulation, or contractile function during low-flow I/R in either the low- or high-fat group, nor did it affect myocardial infarct size. Despite expression of PPAR-alpha in porcine myocardium and effects of fenofibrate on systemic metabolism, treatment with this PPAR-alpha activator does not alter myocardial metabolic or contractile responses to I/R in pigs.

Simultaneous quantification of human cardiac alpha- and beta-myosin heavy chain proteins by MALDI-TOF mass spectrometry.

We have developed a novel method for quantifying protein isoforms, in both relative and absolute terms, based on MALDI-TOF mass spectrometry. The utility of the approach is demonstrated by quantifying the alpha and beta protein isoforms of myosin heavy chain (MyHC) in human atrial tissue. Alpha-MyHC (726-741) and beta-MyHC (724-739) were identified as isoform-specific tryptic peptides. A calibration curve was constructed by plotting ion current ratios against molar ratios of the two peptides prepared synthetically. MyHC was digested by trypsin and the ion current ratio determined for the two tryptic peptides. The ion current ratio was converted to the peptide ratio and hence the isoform ratio by reference to the standard curve. The accuracy of the method was confirmed by a comparison between these results and those determined by an established method of MyHC isoform ratio determination. So that the molar ratio could be converted to absolute values, a third peptide, an analogue of the two peptides being measured, was synthesized for use as an internal standard (IS). The measured ion current ratios of synthetic alpha-MyHC (726-741), beta-MyHC (724-739), and IS peptides were used to generate standard curves. A known quantity of the IS was added to the MyHC digests. The measured ion current ratios were converted to the actual quantities of the isoform-specific peptides and hence the actual quantity of each protein isoform by reference to the standard curves. This method is of general applicability, especially when isoform quantification is required.