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Background: Calcineurin is a calcium-regulated phosphatase that plays a major role in cardiac hypertrophy. We previously described that alternative splicing of the calcineurin Aβ (CnAβ) gene generates the CnAβ1 isoform, with a unique C-terminal region that is different from the autoinhibitory domain present in all other CnA isoforms. In skeletal muscle, CnAβ1 is necessary for myoblast proliferation and stimulates regeneration, reducing fibrosis and accelerating the resolution of inflammation. Its role in the heart is currently unknown.Methods and Results: We generated transgenic mice overexpressing CnAβ1 in postnatal cardiomyocytes under the control of the α-myosin heavy chain promoter. In contrast to previous studies using an artificially truncated calcineurin, CnAβ1 overexpression did not induce cardiac hypertrophy. Moreover, transgenic mice showed improved cardiac function and reduced scar formation after myocardial infarction, with reduced neutrophil and macrophage infiltration and decreased expression of proinflammatory cytokines. Immunoprecipitation and Western blot analysis showed interaction of CnAβ1 with the mTOR complex 2 and activation of the Akt/SGK cardioprotective pathway in a PI3K-independent manner. In addition, gene expression profiling revealed that CnAβ1 activated the transcription factor ATF4 downstream of the Akt/mTOR pathway to promote the amino acid biosynthesis program, to reduce protein catabolism, and to induce the antifibrotic and antiinflammatory factor growth differentiation factor 15, which protects the heart through Akt activation.Conclusions: Calcineurin Aβ1 shows a unique mode of action that improves cardiac function after myocardial infarction, activating different cardioprotective pathways without inducing maladaptive hypertrophy. These features make CnAβ1 an attractive candidate for the development of future therapeutic approaches. [ABSTRACT FROM AUTHOR]

Pathogenic variants in genes that cause dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM) convey high risks for the development of heart failure through unknown mechanisms. Using single-nucleus RNA sequencing, we characterized the transcriptome of 880,000 nuclei from 18 control and 61 failing, nonischemic human hearts with pathogenic variants in DCM and ACM genes or idiopathic disease. We performed genotype-stratified analyses of the ventricular cell lineages and transcriptional states. The resultant DCM and ACM ventricular cell atlas demonstrated distinct right and left ventricular responses, highlighting genotype-associated pathways, intercellular interactions, and differential gene expression at single-cell resolution. Together, these data illuminate both shared and distinct cellular and molecular architectures of human heart failure and suggest candidate therapeutic targets.

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the 'RAF paradox') may have the same effect. BRAF was up-regulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a 'RAF paradox' effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the 'RAF paradox'. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function., (© 2022 The Author(s).)

Acetaminophen ( N -acetyl- p -aminophenol; APAP) toxicity is a common cause of liver damage. In the mouse model of APAP-induced liver injury (AILI), interleukin 11 (IL11) is highly up-regulated and administration of recombinant human IL11 (rhIL11) has been shown to be protective. Here, we demonstrate that the beneficial effect of rhIL11 in the mouse model of AILI is due to its inhibition of endogenous mouse IL11 activity. Our results show that species-matched IL11 behaves like a hepatotoxin. IL11 secreted from APAP-damaged human and mouse hepatocytes triggered an autocrine loop of NADPH oxidase 4 (NOX4)-dependent cell death, which occurred downstream of APAP-initiated mitochondrial dysfunction. Hepatocyte-specific deletion of Il11 receptor subunit alpha chain 1 ( Il11ra1 ) in adult mice protected against AILI despite normal APAP metabolism and glutathione (GSH) depletion. Mice with germline deletion of Il11 were also protected from AILI, and deletion of Il1ra1 or Il11 was associated with reduced c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation and quickly restored GSH concentrations. Administration of a neutralizing IL11RA antibody reduced AILI in mice across genetic backgrounds and promoted survival when administered up to 10 hours after APAP. Inhibition of IL11 signaling was associated with the up-regulation of markers of liver regenerations: cyclins and proliferating cell nuclear antigen (PCNA) as well as with phosphorylation of retinoblastoma protein (RB) 24 hours after AILI. Our data suggest that species-matched IL11 is a hepatotoxin and that IL11 signaling might be an effective therapeutic target for APAP-induced liver damage., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Dilated cardiomyopathy (DCM) is genetically heterogeneous, with >100 purported disease genes tested in clinical laboratories. However, many genes were originally identified based on candidate-gene studies that did not adequately account for background population variation. Here we define the frequency of rare variation in 2538 patients with DCM across protein-coding regions of 56 commonly tested genes and compare this to both 912 confirmed healthy controls and a reference population of 60 706 individuals to identify clinically interpretable genes robustly associated with dominant monogenic DCM., Methods: We used the TruSight Cardio sequencing panel to evaluate the burden of rare variants in 56 putative DCM genes in 1040 patients with DCM and 912 healthy volunteers processed with identical sequencing and bioinformatics pipelines. We further aggregated data from 1498 patients with DCM sequenced in diagnostic laboratories and the Exome Aggregation Consortium database for replication and meta-analysis., Results: Truncating variants in TTN and DSP were associated with DCM in all comparisons. Variants in MYH7, LMNA, BAG3, TNNT2, TNNC1, PLN, ACTC1, NEXN, TPM1 , and VCL were significantly enriched in specific patient subsets, with the last 2 genes potentially contributing primarily to early-onset forms of DCM. Overall, rare variants in these 12 genes potentially explained 17% of cases in the outpatient clinic cohort representing a broad range of adult patients with DCM and 26% of cases in the diagnostic referral cohort enriched in familial and early-onset DCM. Although the absence of a significant excess in other genes cannot preclude a limited role in disease, such genes have limited diagnostic value because novel variants will be uninterpretable and their diagnostic yield is minimal., Conclusions: In the largest sequenced DCM cohort yet described, we observe robust disease association with 12 genes, highlighting their importance in DCM and translating into high interpretability in diagnostic testing. The other genes analyzed here will need to be rigorously evaluated in ongoing curation efforts to determine their validity as Mendelian DCM genes but have limited value in diagnostic testing in DCM at present. This data will contribute to community gene curation efforts and will reduce erroneous and inconclusive findings in diagnostic testing.

Background: Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global posttranscriptional mechanisms underlying fibroblast-to-myofibroblast conversion in the heart have not been explored., Methods: Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. The integration with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients demonstrates that these posttranscriptional mechanisms are also active in the diseased fibrotic human heart., Results: We generated nucleotide-resolution translatome data during the transforming growth factor β1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation, and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in posttranscriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested the same posttranscriptional regulatory network was underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as Pumilio RNA binding family member 2 (PUM2) and Quaking (QKI) that work in concert to regulate the translation of target transcripts in human diseased hearts. Furthermore, silencing of both PUM2 and QKI inhibits the transition of fibroblasts toward profibrotic myofibroblasts in response to transforming growth factor β1., Conclusions: We reveal widespread translational effects of transforming growth factor β1 and define novel posttranscriptional regulatory networks that control the fibroblast-to-myofibroblast transition. These networks are active in human heart disease, and silencing of hub genes limits fibroblast activation. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure.

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFβ1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFβ1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2.

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: We evaluated strategies for identifying disease-causing variants in genetic testing for dilated cardiomyopathy (DCM)., Methods: Cardiomyopathy gene panel testing was performed in 532 DCM patients and 527 healthy control subjects. Rare variants in 41 genes were stratified using variant-level and gene-level characteristics., Results: A majority of DCM cases and controls carried rare protein-altering cardiomyopathy gene variants. Variant-level characteristics alone had limited discriminative value. Differentiation between groups was substantially improved by addition of gene-level information that incorporated ranking of genes based on literature evidence for disease association. The odds of DCM were increased to nearly 9-fold for truncating variants or high-impact missense variants in the subset of 14 genes that had the strongest biological links to DCM (P <0.0001). For some of these genes, DCM-associated variants appeared to be clustered in key protein functional domains. Multiple rare variants were present in many family probands, however, there was generally only one "driver" pathogenic variant that cosegregated with disease., Conclusion: Rare variants in cardiomyopathy genes can be effectively stratified by combining variant-level and gene-level information. Prioritization of genes based on their a priori likelihood of disease causation is a key factor in identifying clinically actionable variants in cardiac genetic testing.

Aim: Hypertrophic cardiomyopathy (HCM) exhibits genetic heterogeneity that is dominated by variation in eight sarcomeric genes. Genetic variation in a large number of non-sarcomeric genes has also been implicated in HCM but not formally assessed. Here we used very large case and control cohorts to determine the extent to which variation in non-sarcomeric genes contributes to HCM., Methods and Results: We sequenced known and putative HCM genes in a new large prospective HCM cohort (n = 804) and analysed data alongside the largest published series of clinically genotyped HCM patients (n = 6179), previously published HCM cohorts and reference population samples from the exome aggregation consortium (ExAC, n = 60 706) to assess variation in 31 genes implicated in HCM. We found no significant excess of rare (minor allele frequency < 1:10 000 in ExAC) protein-altering variants over controls for most genes tested and conclude that novel variants in these genes are rarely interpretable, even for genes with previous evidence of co-segregation (e.g. ACTN2). To provide an aid for variant interpretation, we integrated HCM gene sequence data with aggregated pedigree and functional data and suggest a means of assessing gene pathogenicity in HCM using this evidence., Conclusion: We show that genetic variation in the majority of non-sarcomeric genes implicated in HCM is not associated with the condition, reinforce the fact that the sarcomeric gene variation is the primary cause of HCM known to date and underscore that the aetiology of HCM is unknown in the majority of patients., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Genetic variation is an important determinant of RNA transcription and splicing, which in turn contributes to variation in human traits, including cardiovascular diseases., Results: Here we report the first in-depth survey of heart transcriptome variation using RNA-sequencing in 97 patients with dilated cardiomyopathy and 108 non-diseased controls. We reveal extensive differences of gene expression and splicing between dilated cardiomyopathy patients and controls, affecting known as well as novel dilated cardiomyopathy genes. Moreover, we show a widespread effect of genetic variation on the regulation of transcription, isoform usage, and allele-specific expression. Systematic annotation of genome-wide association SNPs identifies 60 functional candidate genes for heart phenotypes, representing 20% of all published heart genome-wide association loci. Focusing on the dilated cardiomyopathy phenotype we found that eQTL variants are also enriched for dilated cardiomyopathy genome-wide association signals in two independent cohorts., Conclusions: RNA transcription, splicing, and allele-specific expression are each important determinants of the dilated cardiomyopathy phenotype and are controlled by genetic factors. Our results represent a powerful resource for the field of cardiovascular genetics.

Embryonic stem cells (ESC) have the potential to generate all the cell lineages that form the body. However, the molecular mechanisms underlying ESC differentiation and especially the role of alternative splicing in this process remain poorly understood. Here, we show that the alternative splicing regulator MBNL1 promotes generation of the atypical calcineurin Aβ variant CnAβ1 in mouse ESCs (mESC). CnAβ1 has a unique C-terminal domain that drives its localization mainly to the Golgi apparatus by interacting with Cog8. CnAβ1 regulates the intracellular localization and activation of the mTORC2 complex. CnAβ1 knockdown results in delocalization of mTORC2 from the membrane to the cytoplasm, inactivation of the AKT/GSK3β/β-catenin signaling pathway, and defective mesoderm specification. In summary, here we unveil the structural basis for the mechanism of action of CnAβ1 and its role in the differentiation of mESCs to the mesodermal lineage., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Mutations in sarcomeric and cytoskeletal proteins are a major cause of hereditary cardiomyopathies, but our knowledge remains incomplete as to how the genetic defects execute their effects., Methods and Results: We used cysteine and glycine-rich protein 3, a known cardiomyopathy gene, in a yeast 2-hybrid screen and identified zinc-finger and BTB domain-containing protein 17 (ZBTB17) as a novel interacting partner. ZBTB17 is a transcription factor that contains the peak association signal (rs10927875) at the replicated 1p36 cardiomyopathy locus. ZBTB17 expression protected cardiac myocytes from apoptosis in vitro and in a mouse model with cardiac myocyte-specific deletion of Zbtb17, which develops cardiomyopathy and fibrosis after biomechanical stress. ZBTB17 also regulated cardiac myocyte hypertrophy in vitro and in vivo in a calcineurin-dependent manner., Conclusions: We revealed new functions for ZBTB17 in the heart, a transcription factor that may play a role as a novel cardiomyopathy gene., (© 2015 American Heart Association, Inc.)

The recent discovery of heterozygous human mutations that truncate full-length titin (TTN, an abundant structural, sensory, and signaling filament in muscle) as a common cause of end-stage dilated cardiomyopathy (DCM) promises new prospects for improving heart failure management. However, realization of this opportunity has been hindered by the burden of TTN-truncating variants (TTNtv) in the general population and uncertainty about their consequences in health or disease. To elucidate the effects of TTNtv, we coupled TTN gene sequencing with cardiac phenotyping in 5267 individuals across the spectrum of cardiac physiology and integrated these data with RNA and protein analyses of human heart tissues. We report diversity of TTN isoform expression in the heart, define the relative inclusion of TTN exons in different isoforms (using the TTN transcript annotations available at http://cardiodb.org/titin), and demonstrate that these data, coupled with the position of the TTNtv, provide a robust strategy to discriminate pathogenic from benign TTNtv. We show that TTNtv is the most common genetic cause of DCM in ambulant patients in the community, identify clinically important manifestations of TTNtv-positive DCM, and define the penetrance and outcomes of TTNtv in the general population. By integrating genetic, transcriptome, and protein analyses, we provide evidence for a length-dependent mechanism of disease. These data inform diagnostic criteria and management strategies for TTNtv-positive DCM patients and for TTNtv that are identified as incidental findings., (Copyright © 2015, American Association for the Advancement of Science.)

Background: The compensatory actions of the endogenous natriuretic peptide system require adequate processing of natriuretic peptide pro‐hormones into biologically active, carboxyl‐terminal fragments. Natriuretic peptide pro‐peptide processing is accomplished by corin, a transmembrane serine protease expressed by cardiomyocytes. Brain natriuretic peptide (BNP) processing is inadequate in advanced heart failure and is independently associated with adverse outcomes; however, the molecular mechanisms causing impaired BNP processing are not understood. We hypothesized that the development of endoplasmic reticulum stress in cardiomyocytes in advanced heart failure triggers inositol‐requiring protein 1 (IRE1)‐dependent corin mRNA decay, which would favor a molecular substrate favoring impaired natriuretic peptide pro‐peptide processing., Methods and Results: Two independent samples of hearts obtained from patients with advanced heart failure at transplant demonstrated that corin RNA was reduced as Atrial natriuretic peptide (ANP)/BNP RNA increased. Increases in spliced X‐box protein 1, a marker for IRE1‐endoribonuclease activity, were associated with decreased corin RNA. Moreover, ≈50% of the hearts demonstrated significant reductions in corin RNA and protein as compared to the nonfailing control sample. In vitro experiments demonstrated that induction of endoplasmic reticulum stress in cultured cardiomyocytes with thapsigargin activated IRE1's endoribonuclease activity and time‐dependent reductions in corin mRNA. In HL‐1 cells, overexpression of IRE1 activated IRE1 endoribonuclease activity and caused corin mRNA decay, whereas IRE1‐RNA interference with shRNA attenuated corin mRNA decay after induction of endoplasmic reticulum stress with thapsigargin. Pre‐treatment of cells with Actinomycin D to inhibit transcription did not alter the magnitude or time course of thapsigargin‐induced corin mRNA decline, supporting the hypothesis that this was the result of IRE1‐mediated corin mRNA degradation., Conclusions: These data support the hypothesis that endoplasmic reticulum stress‐mediated, IRE1‐dependent targeted corin mRNA decay is a mechanism leading to corin mRNA resulting in corresponding corin protein deficiency may contribute to the pathophysiology of impaired natriuretic peptide pro‐hormone processing in humans processing in humans with advanced systolic heart failure.

Background: Rejection is the major obstacle to survival after cardiac transplantation. We investigated whether overexpression of heat shock protein (Hsp)-27 in mouse hearts protects against acute rejection and the mechanisms of such protection., Methods: Hearts from B10.A mice overexpressing human Hsp-27 (Hsp-27tg), or Hsp-27-negative hearts from littermate controls (LCs) were transplanted into allogeneic C57BL/6 mice. The immune response to B10.A hearts was investigated using quantitative polymerase chain reaction for CD3+, CD4+, CD8+ T cells, and CD14+ monocytes and cytokines (interferon-γ, interleukin [IL]-2, tumor necrosis factor-α, IL-1β, IL-4, IL-5, IL-10, transforming growth factor-β) in allografts at days 2, 5, and 12 after transplantation. The effect of Hsp-27 on ischemia-induced caspase activation and immune activation was investigated., Results: Survival of Hsp-27tg hearts (35±10.37 days, n=10) was significantly prolonged compared with LCs (13.6±3.06 days, n=10, P=0.0004). Hsp-27tg hearts expressed significantly more messenger RNA (mRNA) markers of CD14+ monocytes at day 2 and less mRNA markers of CD3+ and CD8+T cells at day 5 compared with LCs. There was more IL-4 mRNA in Hsp-27tg hearts at day 2 and less interferon-γ mRNA at day 5 compared with LCs. Heat shock protein-27tg hearts subjected to ischemia or to 24 hr ischemia-reperfusion injury demonstrated significantly less apoptosis and activation of caspases 3, 9, and 1 than LCs. T cells removed from C57BL/6 recipients of Hsp-27tg hearts produced a vigorous memory response to B10.A antigens, suggesting immune activation was not inhibited by Hsp-27., Conclusion: Heat shock protein-27 delays allograft rejection, by inhibiting tissue damage, through probably an antiapoptotic pathway. It may also promote an anti-inflammatory subset of monocytes.

Follistatins are extracellular inhibitors of the TGF-β family ligands including activin A, myostatin and bone morphogenetic proteins. Follistatin-like 3 (FSTL3) is a potent inhibitor of activin signalling and antagonises the cardioprotective role of activin A in the heart. FSTL3 expression is elevated in patients with heart failure and is upregulated in cardiomyocytes by hypertrophic stimuli, but its role in cardiac remodelling is largely unknown. Here, we show that the production of FSTL3 by cardiomyocytes contributes to the paracrine activation of cardiac fibroblasts, inducing changes in cell adhesion, promoting proliferation and increasing collagen production. We found that FSTL3 is necessary for this response and for the induction of cardiac fibrosis. However, full activation requires additional factors, and we identify connective tissue growth factor as a FSTL3 binding partner in this process. Together, our data unveil a novel mechanism of paracrine communication between cardiomyocytes and fibroblasts that may provide potential as a therapeutic target in heart remodelling.

Left ventricular mass (LVM) is a highly heritable trait and an independent risk factor for all-cause mortality. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood. Unbiased systems genetics approaches in the rat now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function), interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.

Rationale: Telethonin (also known as titin-cap or t-cap) is a 19-kDa Z-disk protein with a unique β-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonin's in vivo function., Objective: Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation., Methods and Results: By using a variety of different genetically altered animal models and biophysical experiments we show that contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin-titin cross-links via α-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the proapoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis ("mechanoptosis"). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect that may contribute to enhanced rates of apoptosis found in these hearts., Conclusions: Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect that may also play a role in human heart failure.

Combined left ventricular assist device (LVAD) support and pharmacological management of the failing heart can induce reversal of maladaptive cardiac remodelling leading to normalisation of cardiac structure and recovery of cardiac function. The purpose of this study was to compare the gene expression profiles of recovered and non-recovered LVAD patients in order to identify mechanisms underlying the recovery process and differences which may determine outcome. Myocardial expression of 54 genes chosen for their potential role in heart failure and tissue repair was measured using quantitative PCR at the time of LVAD implantation and again at explantation (recovery, n = 13) or transplantation (non-recovery, n = 5). Patients who went on to recover had higher levels of Giα2, EPAC2 and lower levels of IGF2 at the time of LVAD implant compared to patients who failed to recover. During recovery, expression of BNP, IL-1β, VWF and SFRP1 was decreased whilst RGS4 increased. Expression of IGF1 and pro-fibrotic genes was coordinated during recovery. Correlation analysis identified a novel co-regulation of SFRP1 and βMHC in myocardium. In summary, the gene expression profile underlying recovery is complex and comprises both regression and exacerbation of elements of the pathological gene program. Modulation of Giα2, EPAC2, RGS4 and SFRP1 indicates that inhibition of cAMP signalling may potentiate recovery prior to treatment whilst enhanced cAMP and Wnt signalling may underlie recovery during LVAD support.

The β(2)-selective adrenoreceptor agonist clenbuterol promotes both skeletal and cardiac muscle hypertrophy and is undergoing clinical trials in the treatment of muscle wasting and heart failure. We have previously demonstrated that clenbuterol induces a mild physiological ventricular hypertrophy in vivo with normal contractile function and without induction of α-skeletal muscle actin (αSkA), a marker of pathological hypertrophy. The mechanisms of this response remain poorly defined. In this study, we examine the direct action of clenbuterol on cardiocyte cultures in vitro. Clenbuterol treatment resulted in increased cell size of cardiac myocytes with increased protein accumulation and myofibrillar organisation characteristic of hypertrophic growth. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed elevated mRNA expression of ANP and brain natriuretic peptide (BNP) but without change in αSkA, consistent with physiological hypertrophic growth. Clenbuterol-treated cultures also showed elevated insulin-like growth factor I (IGF-1) mRNA and activation of the protein kinase Akt. Addition of either IGF-1 receptor-blocking antibodies or LY294002 in order to inhibit phosphatidylinositol 3-kinase, a downstream effector of the IGF-1 receptor, inhibited the hypertrophic response indicating that IGF-1 signalling is required. IGF-1 expression localised primarily to the minor population of cardiac fibroblasts present in the cardiocyte cultures. Together these data show that clenbuterol acts to induce mild cardiac hypertrophy in cardiac myocytes via paracrine signalling involving fibroblast-derived IGF-1.

Elevated levels of the cardiac transcription factor Hand1 have been reported in several adult cardiac diseases but it is unclear whether this change is itself maladaptive with respect to heart function. To test this possibility, we have developed a novel, inducible transgenic system, and used it to overexpress Hand1 in adult mouse hearts. Overexpression of Hand1 in the adult mouse heart leads to mild cardiac hypertrophy and a reduction in life expectancy. Treated mice show no significant fibrosis, myocyte disarray or congestive heart failure, but have a greatly reduced threshold for induced ventricular tachycardia, indicating a predisposition to cardiac arrhythmia. Within 48 h, they show a significant loss of connexin43 protein from cardiac intercalated discs, with increased intercalated disc beta-catenin expression at protein and RNA levels. These changes are sustained during prolonged Hand1 overexpression. We propose that cardiac overexpression of Hand1 offers a useful mouse model of arrhythmogenesis and elevated HAND1 may provide one of the molecular links between the failing heart and arrhythmia.

Clenbuterol is currently being used as part of a clinical trial into a novel therapeutic approach for the treatment of end-stage heart failure. The purpose of this study was to determine the global pattern of myocardial gene expression in response to clenbuterol and to identify novel targets and pathways involved. Rats were treated with clenbuterol (n = 6) or saline (n = 6) for periods of 1, 3, 9, or 28 days. Rats treated for 28 days were also subject to continuous electrocardiogram analysis using implantable telemetry. RNA was extracted from rats at days 1 and 28 and used from microarray analysis, and further samples from rats at days 1, 3, 9, and 28 were used for analysis by real-time polymerase chain reaction. Clenbuterol treatment induced rapid development of cardiac hypertrophy with increased muscle mass at day 1 and elevated heart rate and QT interval throughout the 28-day period. Microarray analysis revealed a marked but largely transitory change in gene expression with 1,423 genes up-regulated and 964 genes down-regulated at day 1. Up-regulated genes revealed an unexpected association with angiogenesis and integrin-mediated cell adhesion and signaling. Moreover, direct treatment of endothelial cells cultured in vitro resulted in increased cell proliferation and tube formation. Our data show that clenbuterol treatment is associated with rapid cardiac hypertrophy and identify angiogenesis and integrin signaling as novel pathways of clenbuterol action. The data have implications both for our understanding of the physiologic hypertrophy induced by clenbuterol and for treatment of heart failure.

Background: Abnormalities in the extracellular matrix (ECM) can occur in heart failure. In this study we analyzed ECM gene expression in patients with advanced dilated cardiomyopathy who did and did not develop sustained myocardial recovery after left ventricular assist device (LVAD) unloading combined with pharmacologic therapy., Methods: Myocardial gene expression of collagens (COL1A1 and COL3A1), fibronectin (FN), matrix metalloproteinases (MMPs 1 to 14), tissue inhibitors of metalloproteinases (TIMPs 1 to 4), connective tissue growth factor (CTGF), transforming growth factor-beta1 (TGF-beta1) and THY1 was measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) at LVAD implantation and again at explantation (recovery, n = 11) or transplantation (non-recovery, n = 5)., Results: The non-recovery group had higher levels of pro-fibrotic markers (COL1A1, TGF-beta1 and THY1) at implantation compared with the recovery group (1.82 +/- 0.74-, 1.81 +/- 0.69- and 3.01 +/- 1.70-fold, respectively; p

Follistatins play roles in diverse biological processes including cell proliferation, wound healing, inflammation, and skeletal muscle growth, yet their role in the heart is currently unknown. We have investigated the myocardial expression profile and cellular distribution of follistatin (FST) and the FST-like genes FSTL1 and FSTL3 in the normal and failing heart. Expression was further analyzed in the novel setting of recovery from heart failure in myocardium obtained from patients who received combined mechanical (left ventricular assist device) and pharmacological therapy. Real-time PCR revealed that FSTL1 and FSTL3 expression was elevated in heart failure but returned to normal after recovery. FSTL3 expression levels correlated with molecular markers of disease severity and FSTL1 with the endothelial cell marker CD31, suggesting a potential link with vascularization. FSTL1 levels before treatment correlated with cardiac function after recovery, suggesting initial levels may influence long-term outcome. Immunohistochemistry revealed that FST was primarily localized to fibroblasts and vascular endothelium within the heart, whereas FSTL1 was localized to myocytes, endothelium, and smooth muscle cells and FSLT3 to myocytes and endothelium. Microarray analysis revealed that FST and FSTL1 were associated with extracellular matrix-related and calcium-binding proteins, whereas FSTL3 was associated mainly with cell signaling and transcription. These data show for the first time that elevated myocardial expression of FST-like genes is a feature of heart failure and may be linked to both disease severity and mechanisms underlying recovery, revealing new insight into the pathogenesis of heart failure and offering novel therapeutic targets.

Background: Intramyocardial injection of skeletal myoblasts (SMB) has been shown to be a promising strategy for treating post-infarction chronic heart failure. However, insufficient therapeutic benefit and occurrence of ventricular arrhythmias are concerns. We hypothesised that the use of a retrograde intracoronary route for SMB-delivery might favourably alter the behaviour of the grafted SMB, consequently modulating the therapeutic effects and arrhythmogenicity., Methods and Results: Three weeks after coronary artery ligation in female wild-type rats, 5x10(6) GFP-expressing SMB or PBS only (control) were injected via either the intramyocardial or retrograde intracoronary routes. Injection of SMB via either route similarly improved cardiac performance and physical activity, associated with reduced cardiomyocyte-hypertrophy and fibrosis. Grafted SMB via either route were only present in low numbers in the myocardium, analysed by real-time PCR for the Y-chromosome specific gene, Sry. Cardiomyogenic differentiation of grafted SMB was extremely rare. Continuous ECG monitoring by telemetry revealed that only intramyocardial injection of SMB produced spontaneous ventricular tachycardia up to 14 days, associated with local myocardial heterogeneity generated by clusters of injected SMB and accumulated inflammatory cells. A small number of ventricular premature contractions with latent ventricular tachycardia were detected in the late-phase of SMB injection regardless of the injection-route., Conclusion: Retrograde intracoronary injection of SMB provided significant therapeutic benefits with attenuated early-phase arrhythmogenicity in treating ischaemic cardiomyopathy, indicating the promising utility of this route for SMB-delivery. Late-phase arrhythmogenicity remains a concern, regardless of the delivery route.

Side population cells have been found in various types of adult tissue including heart and are presumed to be tissue-specific stem/progenitor cells. In the present study, we confirmed the presence of cardiac side population (cSP) cells, which showed both the Hoechst 33342 efflux ability and ABCG2 expression, in adult murine heart. Flow cytometric analysis showed that more than half of cSP cells expressed the endothelial marker VE-cadherin or the smooth muscle markers, alpha-smooth muscle actin and desmin. In addition, immunohistochemical analysis demonstrated that ABCG2(+) cells were mainly localized within vascular walls. Quantitative RT-PCR analysis demonstrated that VE-cadherin(-) cSP cells progressively expressed Nkx2.5 and cardiac troponin T with time in culture. VE-cadherin(-) cSP cells also expressed mesodermal-mesenchymal-associated markers and differentiated into osteocytes and adipocytes. These results highlight the heterogeneic nature of cSP cells, consisting of vascular endothelial cells, smooth muscle cells, and mesenchymal stem/progenitor cells including potential cardiomyogenic cells.

Aims: Combined left ventricular assist device (LVAD) and pharmacological therapy has been proposed to favour myocardial recovery in patients with end-stage heart failure (HF). Clenbuterol (Clen), a beta(2)-adrenoceptor (beta(2)-AR) agonist, has been used as a part of this strategy. In this study, we investigated the direct effects of clenbuterol on unloaded myocardium in HF., Methods and Results: Left coronary artery ligation or sham operation was performed in male Lewis rats. After 4-6 weeks, heterotopic abdominal transplantation of the failing hearts into normal recipients was performed to induce LV unloading (UN). Recipient rats were treated with saline (Sal) or clenbuterol (2 mg/kg/day) via osmotic minipumps (HF + UN + Sal or HF + UN + Clen) for 7 days. Non-transplanted HF animals were treated with Sal (Sham + Sal, HF + Sal) or clenbuterol (HF + Clen). LV myocytes were isolated and studied using optical, fluorescence, and electrophysiological techniques. Clenbuterol treatment improved in vivo LV function measured with echocardiography (LVEF (%): HF 35.9 +/- 2 [16], HF + Clen 52.1 +/- 1.4 [16]; P < 0.001; mean +/- SEM [n]). In combination with unloading, clenbuterol increased sarcomere shortening (amplitude (microm): HF + UN + Clen 0.1 +/- 0.01 [50], HF + UN + Sal 0.07 +/- 0.01 [38]; P < 0.001) by normalizing the depressed myofilament sensitivity to Ca(2+) (slope of the linear relationship between Ca(2+) transient and sarcomere shortening hysteresis loop during relaxation (microm/ratio unit): HF + UN + Clen 2.13 +/- 0.2 [52], HF + UN + Sal 1.42 +/- 0.13 [38]; P < 0.05)., Conclusion: Clenbuterol treatment of failing rat hearts, alone or in combination with mechanical unloading, improves LV function at the whole-heart and cellular levels by affecting cell morphology, excitation-contraction coupling, and myofilament sensitivity to calcium. This study supports the use of this drug in the strategy to enhance recovery in HF patients treated with LVADs and also begins to elucidate some of the possible cellular mechanisms responsible for the improvement in LV function.

Aims: A novel combination therapy consisting of a left ventricular assist device (LVAD) combined with pharmacologic therapy including the selective beta(2)-agonist, clenbuterol, has shown promise in restoring ventricular function in patients with heart failure. The aim of this study was to identify common genes and signalling pathways whose expression was associated with reversal of heart failure and restoration of ventricular function., Methods and Results: Microarray analysis was performed on six paired human heart samples harvested at the time of LVAD implant and at the time of LVAD explant for recovery of ventricular function (post). Follow-up data shows that the improvements in ventricular function have been maintained for an average of 3.8 years post-explant. Analysis of the gene expression data revealed: (i) a significant association of integrin pathway signalling with recovery and (ii) the identification of several novel targets including, EPAC2, in the well-described cAMP pathway whose expression was down-regulated with recovery, and was associated with improvements in cardiac contractility, metabolism, and function., Conclusion: This data set represents the first description of signalling pathways associated with the functional recovery of end-stage human heart failure and the identification of new targets in the human heart that are modified by this combination therapy.

Alternatively spliced endothelin (ET-1) receptor transcripts have been identified, but their significance to the functional effects of ET-1 has not been established. We have investigated the presence and influence of alternatively spliced ET(A) receptor transcripts on ET-1 mediated contraction of segments of human saphenous vein. The expression of ET(A) receptor transcripts was examined with quantitative reverse transcription-polymerase chain reaction (qPCR) studies, while the response of veins to ET-1 was tested with in vitro organ bath techniques. The expression of four different transcripts for the ET(A) receptor, in which either exon 3 is spliced out (Delta3), exon 4 is spliced out (Delta4), both 3 and 4 spliced out (Delta3,4) and when both exons 2 and 4 (Delta2,4) are spliced out were identified. Functional studies showed that a lack of efficacy and potency of ET-1 is associated with a significantly lower expression of the Delta3,4 transcript. ET(A) receptor antagonism was insurmountable in samples that had lower levels of the Delta3,4 transcript, while samples from patients with higher expression of the Delta3,4 showed surmountable antagonism with BQ123. These results suggest that there is a genetic basis for the variability between individuals for the contractile effect of ET-1 at ET(A) receptors.

Background: Mechanisms underlying the rapid deterioration of heart failure patients who subsequently require left ventricular assist device (LVAD) support are poorly understood. Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) play a key role in myocardial remodelling and heart failure. We hypothesized that MMP and TIMP expression would be altered in these patients., Methods: Quantitative polymerase chain reaction was used to measure myocardial messenger RNA levels of MMP1 to MMP14, TIMP1 to TIMP4, collagen I and collagen III in 24 dilated cardiomyopathy (DCM) patients with deteriorating clinical status who required LVAD support (LVAD Group) and in 7 stable DCM patients undergoing transplantation without need for LVAD support (Tx Group)., Results: Levels of MMP1, MMP8 and TIMP4 were higher in the LVAD Group compared with the Tx Group (188% +/- 141%, 646% +/- 432%, and 66% +/- 33% higher, respectively, p < 0.05) whereas MMP2, MMP9, MMP10, MMP11, and MMP14 levels were similar. MMP3, MMP7, MMP12, and MMP13 were undetectable. All TIMPs were generally higher in the LVAD group, but only TIMP4 reached significance. Collagen I and III were not altered. We tested for correlations between MMP and TIMP expression with myocardial cytokine levels. MMP8 correlated positively with interleukin-6 and interleukin-1beta, suggesting a link between cytokines and MMPs in these patients., Conclusions: The data show that high myocardial collagenase (MMP1 and MMP8) expression without compensatory changes in collagen or TIMP expression is a feature of patients requiring LVAD support. This may be linked in part to elevated cytokine expression and suggests collagenase activity may be an important therapeutic target in deteriorating heart failure.

The real-time quantitative polymerase chain reaction (PCR), an increasingly popular technique for the detection of DNA, combines a high degree of accuracy with extreme sensitivity. In this chapter we describe the use of real-time quantitative PCR in transplantation research in two areas in which this method is commonly applied: the accurate quantification of mRNA in tissue samples and genotyping of DNA. These are described in the context of cardiac transplantation, but they are of equal relevance to other areas of transplant biology.

Cardiac hypertrophy and heart failure occur in association to alterations in glucose uptake and metabolism. Phenylephrine, among other hypertrophic agonists, has been reported to increase expression of GLUT1 in neonatal rat cardiac myocytes by activating transcription. However, the specific cis- or trans-acting factors in the GLUT1 gene that are targeted by this agonist remain elusive. Here we describe that the activity of the -99/+134 basal promoter of rat GLUT1 is increased by phenylephrine. Nevertheless, this is not mediated by previously described binding sites (GC-box, MG1E) in the promoter. Rather, the TATA box is required by the agonist to activate transcription from the promoter. Interestingly, The Ras-ERK mitogen-activated protein (MAP) kinase pathway is involved in the actions of phenylephrine on GLUT1 transcription, and the effects of Ras on the activity of the promoter depend on the integrity of the TATA box. Our data indicate that phenylephrine induces the expression of the TBP-associated factor TAF(II)250 mRNA, which increases in parallel to the expression of GLUT1, suggesting that altering the expression of basal transcription factors could be one mechanism by which phenylephrine may regulate the activity of the GLUT1 promoter.

Background: The expression of the "protective" genes A20, heme oxygenase (HO)-1, and Bcl-xl in rodent allografts and xenografts correlates with long-term survival of transplanted hearts. We investigated the expression of HO-1, Bcl-2, and A20 in sequential biopsies from nine cardiac transplant recipients by using quantitative real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry., Methods: Five to 16 endomyocardial biopsies were analyzed from each patient 7 to 365 days after transplantation. Biopsies were classified as acute rejection (AR) by International Society of Heart and Lung Transplantation criteria. mRNA values were normalized against an endogenous control gene (18S), and protein expression was analyzed by immunohistochemistry., Results: All genes were expressed at every time point. HO-1 was significantly higher in the first 2 months (2 months vs. 10+ months, P<0.05) and was associated with AR (0.30+/-0.07) versus nonrejection (0.16+/-0.02, P=0.026). In contrast, expression of Bcl-2 and A20 was low at 2 months, but both increased with time (P<0.05, 2 months vs. 10+ months for Bcl-2 and A20). There was no significant association of Bcl-2 or A20 with AR. Immunocytochemistry revealed that HO-1 localizes to infiltrating cells and not parenchymal cells in cardiac biopsies. In contrast, Bcl-2 and A20 were found to localize to endothelial, smooth muscle, and infiltrating cells., Conclusions: HO-1 is induced early after transplantation, whereas Bcl-2 and A20 seem to be induced as part of the chronic response. These differences together with different localization sites in vivo suggest they have different roles in protection from injury after cardiac transplantation.

The proteins, spectrin and 4.1 confer support and resilience to animal cell membranes, and promote assembly of multimeric, membrane-bound signalling complexes. Protein 4.1 also plays important roles in tumour suppression and the regulation of cell proliferation. To assess relative tissue expression of the four genes encoding human protein 4.1, we measured mRNA levels using quantitative real-time polymerase chain reaction. We compared 4.1 expression with that of a major splice variant of spectrin, betaIISigma2 that has a shortened C-terminus lacking a pleckstrin homology domain. mRNA for 4.1R is four-fold higher in bone marrow than in tissues with the next highest prevalence: cerebellum, lung, testis and thymus. 4.1G mRNA is highly expressed in brain, spinal cord and testis; 4.1N in brain, spinal cord and adrenal gland; 4.1B in testis, brain, spinal cord, and kidney. Thus, 4.1N, 4.1B and 4.1G all show high accumulation in nervous tissues. mRNA for betaIISigma2-spectrin is ubiquitous, but most abundant in cardiac and nervous tissues. Comparative transcript abundance was analysed in heart and brain. betaIISigma2-spectrin was the most abundant transcript in heart with levels 5 fold greater than 4.1G or 4.1N and at least 9 fold greater than 4.1B. In brain, 4.1N was the most abundant transcript, with levels 2.4 fold greater than 4.1B and at least 4 fold greater than 4.1G or betaIISigma2-spectrin. 4.1R abundance was very low in both tissues. Whilst we expected that 4.1 mRNAs would feature highly in muscle and nerve, we note their high abundance in testis, indicating previously unsuspected functions in reproduction.

Cardiac muscle development is characterised by the activation of contractile protein genes and subsequent modulation of expression resulting, ultimately, in the formation of a mature four-chambered organ. Myocardial gene expression is also altered in the adult in response to pathological stimuli and this is thought to contribute to the altered contractile characteristics of the diseased heart. We have examined the expression of the slow skeletal troponin T (TnT) gene in the human heart during development and in disease using whole mount in situ hybridisation and real-time quantitative (TaqMan) polymerase chain reaction (PCR). Slow skeletal TnT mRNA shows transitory and regional expression in the early foetal heart, which occurs at different times in atria and ventricles. In ventricular myocardium, expression is seen in the outer epicardial layer at a time when the coronary circulation is being established. Expression was detected at low levels in the adult human heart and was significantly increased in end-stage heart failure. Similarly, expression was readily detectable during early rat heart development and was up-regulated in pressure overload hypertrophy in adult. Together these data show for the first time that slow skeletal TnT mRNA is readily detectable during early human heart development. They further suggest that slow skeletal TnT may be responsive to myocardial stress and that elevated levels may contribute to myocardial dysfunction in adult disease.

Background: Cytokine activation in the myocardium of deteriorating patients with heart failure who undergo left ventricular assist-device (LVAD) implantation has been documented, but the underlying mechanisms remain poorly understood. We hypothesized the innate immune system is activated with expression of Toll-like receptor 4 (TLR4), leading to cytokine activation in these patients., Methods: We used quantitative real-time reverse-transcriptase polymerase chain reaction to measure TLR4, interleukin-1 (IL-1) receptor, IL-1 beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) mRNA expression in myocardial samples from 36 patients. We compared 18 patients who underwent LVAD implantation with 18 patients with less severe heart failure who underwent elective heart transplantation., Results: Toll-like receptor 4 expression was 1.69-fold greater (p < 0.05) and IL-1 receptor expression was 3.64-fold greater (p < 0.0001) in the deteriorating patients who required LVADs. Myocardial TNF-alpha (1.71-fold, p < 0.05), IL-6 (2.57-fold, p < 0.005), and IL-1 beta (9.78-fold, p < 0.001) also were increased in the LVAD candidates. Toll-like receptor 4 expression correlated strongly with IL-1 receptor expression (r= 0.75, p < 0.0001) and with IL-1 beta expression in individual patients (r = 0.7, p < 0.0001). Interleukin-1 receptor expression also correlated with IL-1 beta expression (r = 0.78, p < 0.0001) within patients. We found no correlation between TLR4 and either TNF-alpha or IL-6 expression., Conclusions: Patients who required LVAD support showed evidence of innate immune system activation, indicated by an increase in the key effector molecule TLR4 associated with a specific pattern of cytokine expression in the myocardium.

Background: The authors previously identified and compared alterations in gene expression in the myocardia of patients with deteriorating heart failure who underwent left ventricular assist device (LVAD) implantation with those of patients with stable end-stage failure (ESF). We hypothesized that matrix metalloproteinases (MMPs) and their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs), would be implicated in the mechanisms that underlie deteriorating heart failure., Methods: Gridded macro-array filters were used to provide a broad overview of MMP and TIMP mRNA expression in heart failure. Precise mRNA levels of TIMP1, MMP1, and beta-spectrin were determined using quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) of myocardial samples from 27 patients with deteriorating heart failure who underwent LVAD implantation, from 17 patients with stable ESF who underwent elective heart transplantation, and from 28 donor organs with good hemodynamic function., Results: Gridded macro-arrays analysis of pooled failing heart samples determined that TIMP1 mRNA was the most readily detectable TIMP in failing myocardium. Quantitative RT-PCR showed that expression levels in individual patients were similar in patients with stable ESF (1.00 +/- 0.24, n = 17) and in donor organ samples (1.49 +/- 0.22, n = 28) but were significantly increased in the deteriorating heart failure group (5.38 +/- 0.32, n = 26, p < 0.0001 compared with patients with ESF). Similarly, MMP1 levels did not differ between donor and ESF groups but increased in the deteriorating failure group (6.04 +/- 0.50, n = 27, p < 0.001 compared with the ESF group). Levels of beta-II spectrin were the same in all 3 groups. Both TIMP1 and MMP1 showed positive correlation with each other and with previously determined levels of mRNA for both interleukin-1beta (IL-1beta) and IL-6 in this patient series when considering all patients individually, but neither correlated with tumor necrosis factor alpha., Conclusions: Patients with deteriorating heart failure have increased expression of TIMP1 and MMP1 mRNA. Correlation with pro-inflammatory cytokines suggests common pathways of regulation and potential activation by IL-6 and IL1-beta.

Background: Molecular mechanisms underlying the deterioration of patients undergoing LV assist device (LVAD) implantation remain poorly understood. We studied the cytokines tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta and IL-6 and the terminal stage of the apoptotic pathway in patients with decompensating heart failure who required LVAD support and compared them with patients with less severe heart failure undergoing elective heart transplantation., Methods and Results: Myocardial and serum samples from 23 patients undergoing LVAD implantation were compared with those from 36 patients undergoing elective heart transplantation. Myocardial TNF-alpha mRNA (1.71-fold; P<0.05) and protein (3.43+/-0.19 versus 2.95+/-0.10 pg/mg protein; P<0.05) were elevated in the LVAD patients. Immunocytochemistry demonstrated TNF expression in the myocytes. Serum TNF-alpha was also elevated (12.5+/-1.9 versus 4.0+/-0.4 pg/mL; P<0.0001) in the LVAD patients. IL-6 mRNA (2.57-fold higher; P<0.005) and protein (27.83+/-9.35 versus 4.26+/-1.24 pg/mg protein; P<0.001) were higher in the LVAD candidates, as was serum IL-6 (79.3+/-23.6 versus 7.1+/-1.6 pg/mL; P<0.0001). Interleukin-1beta mRNA expression was 9.78-fold higher in the LVAD patients (P<0.001). iNOS mRNA expression was similar to that in advanced heart failure patients and was not further elevated in the LVAD patients. Levels of procaspase-9 (8.02+/-0.91 versus 6.16+/-0.43 oligodeoxynucleotide [OD] units; P<0.01), cleaved caspase-9 (10.02+/-1.0 versus 7.34+/-0.40 OD units; P<0.05), intact and spliced DFF-45 (4.58+/-0.75 versus 2.84+/-0.23 OD units; P<0.05) were raised in LVAD patients, but caspase-3 and human nuclease CPAN were not., Conclusions: Elevated TNF-alpha, IL-1beta, and IL-6 and alterations in the apoptotic pathway were found in the myocardium and elevated TNF-alpha and IL-6 in serum of deteriorating patients who required LVAD support. These occurrences may have therapeutic implications and influence the timing of LVAD insertion.