Thoracic Aortic Constriction Research Articles

0204: Proteome-wide sex-related differences in response to mouse thoracic aortic constriction: molecular bio-signature of failing hearts

Chronic pressure overload (PO) induces pathological left ventricular hypertrophy (LVH) leading to congestive heart failure (HF). Over-expression of FKBP12.6 (FK506 binding protein (K)) in mice should prevent Ca2+-leak during diastole and may improve overall cardiac function. In order to decipher molecular mechanisms involved in thoracic aortic constriction (TAC)-induced cardiac remodelling and the influence of gender and genotype, we performed a proteomic analysis using 2D-DIGE, mass spectrometry and bioinformatics techniques to identify alterations in characteristic biological networks. Wild type (W) and K mice of both genders underwent TAC. Thirty days post-TAC, the altered cardiac remodelling was accompanied with systolic and diastolic dysfunction in all experimental groups. A gender difference in inflammatory protein expression (fibrinogen, alpha-1-antitrypsin isoforms) and in calreticulin occurred (males > females). Detoxification enzymes and cytoskeletal proteins were noticeably increased in K mice. Both non- and congestive failing mouse heart exhibited down- and up-regulation of proteins related to mitochondrial function and purine metabolism, respectively. HF was characterized by a decrease in enzymes related to iron homeostasis, and altered mitochondrial protein expression related to fatty acid metabolism, glycolysis and redox balance. Moreover, two distinct differential protein profiles characterized TAC-induced pathological LVH and congestive HF in all TAC mice. FKBP12.6 over-expression did not influence TACinduced deleterious effects. Huntingtin was revealed as a potential mediator for HF. A broad dysregulation of signaling proteins associated with congestive HF suggested that different sets of proteins could be selected as useful biomarkers for HF progression and might predict outcome in PO-induced pathological LVH.

Connective Tissue Growth Factor Inhibition Attenuates Left Ventricular Remodeling and Dysfunction in Pressure Overload–Induced Heart Failure

Connective tissue growth factor (CTGF) is involved in the pathogenesis of various fibrotic disorders. However, its role in the heart is not clear. To investigate the role of CTGF in regulating the development of cardiac fibrosis and heart failure, we subjected mice to thoracic aortic constriction (TAC) or angiotensin II infusion, and antagonized the function of CTGF with CTGF monoclonal antibody (mAb). After 8 weeks of TAC, mice treated with CTGF mAb had significantly better preserved left ventricular (LV) systolic function and reduced LV dilatation compared with mice treated with control immunoglobulin G. CTGF mAb-treated mice exhibited significantly smaller cardiomyocyte cross-sectional area and reduced expression of hypertrophic marker genes. CTGF mAb treatment reduced the TAC-induced production of collagen 1 but did not significantly attenuate TAC-induced accumulation of interstitial fibrosis. Analysis of genes regulating extracellular matrix proteolysis showed decreased expression of plasminogen activator inhibitor-1 and matrix metalloproteinase-2 in mice treated with CTGF mAb. In contrast to TAC, antagonizing the function of CTGF had no effect on LV dysfunction or LV hypertrophy in mice subjected to 4-week angiotensin II infusion. Further analysis showed that angiotensin II-induced expression of hypertrophic marker genes or collagens was not affected by treatment with CTGF mAb. In conclusion, CTGF mAb protects from adverse LV remodeling and LV dysfunction in hearts subjected to pressure overload by TAC. Antagonizing the function of CTGF may offer protection from cardiac end-organ damage in patients with hypertension.

Gender-Specific Potential Inhibitory Role of Ca2+/Calmodulin Dependent Protein Kinase Phosphatase (CaMKP) in Pressure-Overloaded Mouse Heart

BackgroundCa2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling.Methodology and Principal FindingsFive week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity.Conclusions/SignificanceGender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts. Therefore, CaMKP could be considered as an important novel cellular target for the development of new therapeutic strategies for heart diseases.

HMGB1 Is Involved in the Protective Effect of the PPARαAgonist Fenofibrate against Cardiac Hypertrophy

High mobility group box 1 (HMGB1) is a ubiquitous nuclear DNA-binding protein whose function is dependent on its cellular location. Extracellular HMGB1 is regarded as a delayed mediator of proinflammatory cytokines for initiating and amplifying inflammatory responses, whereas nuclear HMGB1 has been found to prevent cardiac hypertrophy and heart failure. Because fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, has shown both protective effects against cardiac hypertrophy and inhibitory effects against inflammation, the potential modulation of HMGB1 expression and secretion by fenofibrate is of great interest. We herein provide evidence that fenofibrate modulates basal and LPS-stimulated HMGB1 expression and localization in addition to secretion of HMGB1 in cardiomyocytes. In addition, administration of fenofibrate to mice prevented the development of cardiac hypertrophy induced by thoracic transverse aortic constriction (TAC) while increasing levels of nuclear HMGB1. Altogether, these data suggest that fenofibrate may inhibit the development of cardiac hypertrophy by regulating HMGB1 expression, which provides a new potential strategy to treat cardiac hypertrophy.

Docosahexaenoic Acid Supplementation Alters Key Properties of Cardiac Mitochondria and Modestly Attenuates Development of Left Ventricular Dysfunction in Pressure Overload-Induced Heart Failure

Supplementation with the n3 polyunsaturated fatty acid docosahexaenoic acid (DHA) is beneficial in heart failure patients, however the mechanisms are unclear. DHA is incorporated into membrane phospholipids, which may prevent mitochondrial dysfunction. Thus we assessed the effects of DHA supplementation on cardiac mitochondria and the development of heart failure caused by aortic pressure overload. Pathological cardiac hypertrophy was generated in rats by thoracic aortic constriction. Animals were fed either a standard diet or were supplemented with DHA (2.3% of energy intake). After 14weeks, heart failure was evident by left ventricular hypertrophy and chamber enlargement compared to shams. Left ventricle fractional shortening was unaffected by DHA treatment in sham animals (44.1 ± 1.6% vs. 43.5 ± 2.2% for standard diet and DHA, respectively), and decreased with heart failure in both treatment groups, but to a lesser extent in DHA treated animals (34.9 ± 1.7%) than with the standard diet (29.7 ± 1.5%, P < 0.03). DHA supplementation increased DHA content in mitochondrial phospholipids and decreased membrane viscosity. Myocardial mitochondrial oxidative capacity was decreased by heart failure and unaffected by DHA. DHA treatment enhanced Ca(2+) uptake by subsarcolemmal mitochondria in both sham and heart failure groups. Further, DHA lessened Ca(2+)-induced mitochondria swelling, an index of permeability transition, in heart failure animals. Heart failure increased hydrogen peroxide-induced mitochondrial permeability transition compared to sham, which was partially attenuated in interfibrillar mitochondria by treatment with DHA. DHA decreased mitochondrial membrane viscosity and accelerated Ca(2+) uptake, and attenuated susceptibility to mitochondrial permeability transition and development of left ventricular dysfunction.

GW24-e1873 Regulatory mechanism of Tomoregulin-1 in the development of cardiomyopathy

ObjectivesTomoregulin-1 is a growth factor which was found in recent years, and it is reported that gene encoding Tomoregulin-1 mainly involved in embryonic development and adult central nervous system function....

Comparative differential proteomic profiles of nonfailing and failing hearts after in vivo thoracic aortic constriction in mice overexpressing FKBP12.6

Chronic pressure overload (PO) induces pathological left ventricular hypertrophy (LVH) leading to congestive heart failure (HF). Overexpression of FKBP12.6 (FK506-binding protein [K]) in mice should prevent Ca2+-leak during diastole and may improve overall cardiac function. In order to decipher molecular mechanisms involved in thoracic aortic constriction (TAC)-induced cardiac remodeling and the influence of gender and genotype, we performed a proteomic analysis using two-dimensional differential in-gel electrophoresis (2D-DIGE), mass spectrometry, and bioinformatics techniques to identify alterations in characteristic biological networks. Wild-type (W) and K mice of both genders underwent TAC. Thirty days post-TAC, the altered cardiac remodeling was accompanied with systolic and diastolic dysfunction in all experimental groups. A gender difference in inflammatory protein expression (fibrinogen, α-1-antitrypsin isoforms) and in calreticulin occurred (males > females). Detoxification enzymes and cytoskeletal proteins were noticeably increased in K mice. Both non- and congestive failing mouse heart exhibited down- and upregulation of proteins related to mitochondrial function and purine metabolism, respectively. HF was characterized by a decrease in enzymes related to iron homeostasis, and altered mitochondrial protein expression related to fatty acid metabolism, glycolysis, and redox balance. Moreover, two distinct differential protein profiles characterized TAC-induced pathological LVH and congestive HF in all TAC mice. FKBP12.6 overexpression did not influence TAC-induced deleterious effects. Huntingtin was revealed as a potential mediator for HF. A broad dysregulation of signaling proteins associated with congestive HF suggested that different sets of proteins could be selected as useful biomarkers for HF progression and might predict outcome in PO-induced pathological LVH.

Atrial endothelial impairment through Toll-like receptor 4 signaling causes atrial thrombogenesis

Low-grade inflammation associated with heart failure (HF) is known to deteriorate cardioembolic stroke in patients with atrial fibrillation (AF). Little is known about the relationship between atrial endothelial impairment induced by innate immunity and thrombus formation. We examined whether atrial endothelial impairment through Toll-like receptor (TLR) 4 signaling causes atrial thrombogenesis. TLR4, heat shock protein 60, and vascular cell adhesion molecule (VCAM)-1 expression were higher in the atrium of AF patients who underwent valve replacement surgery with HF compared with those without it (p < 0.05). We created thoracic transverse aortic constriction (TAC) in TLR4 knock-out (KO) and wild-type (WT) mice. Atrial thrombosis was observed less frequently in TLR4 KO mice (4/15) than in WT mice (16/20) 4 weeks after TAC despite similar severity of heart failure. The decrease in endothelial nitric oxide synthase (eNOS) phosphorylation and increase in VCAM-1 and plasminogen activator inhibitor (PAI)-1 expression, observed in the atrium of WT mice following TAC, were significantly attenuated in TLR4 KO mice (p < 0.05). Nuclear factor-κB (NF-κB) activation after TAC was attenuated in TLR4 KO mice compared with WT mice. Activation of mitogen-activated protein kinase p38 (p38) after TAC was also attenuated in TLR4 KO mice (p < 0.05). Thus, increased VCAM-1 and PAI-1, and decreased eNOS phosphorylation through the TLR4/NFκB/p38 pathway, may be associated with atrial thrombogenesis in the heart failure mice model. Atrial endothelial impairment through the TLR4 signaling may play a role in atrial thrombogenesis in AF patients with HF.

Cardiac nuclear high mobility group box 1 prevents the development of cardiac hypertrophy and heart failure

AimsHigh mobility group box 1 (HMGB1) is an abundant and ubiquitous nuclear DNA-binding protein that has multiple functions dependent on its cellular location. HMGB1 binds to DNA, facilitating numerous nuclear functions including maintenance of genome stability, transcription, and repair. However, little is known about the effects of nuclear HMGB1 on cardiac hypertrophy and heart failure. The aim of this study was to examine whether nuclear HMGB1 plays a role in the development of cardiac hypertrophy induced by pressure overload.Methods and resultsAnalysis of human biopsy samples by immunohistochemistry showed decreased nuclear HMGB1 expression in failing hearts compared with normal hearts. Nuclear HMGB1 decreased in response to both endothelin-1 (ET-1) and angiotensin II (Ang II) stimulation in neonatal rat cardiomyocytes, where nuclear HMGB1 was acetylated and translocated to the cytoplasm. Overexpression of nuclear HMGB1 attenuated ET-1 induced cardiomyocyte hypertrophy. Thoracic transverse aortic constriction (TAC) was performed in transgenic mice with cardiac-specific overexpression of HMGB1 (HMGB1-Tg) and wild-type (WT) mice. Cardiac hypertrophy after TAC was attenuated in HMGB1-Tg mice and the survival rate after TAC was higher in HMGB1-Tg mice than in WT mice. Induction of foetal cardiac genes was decreased in HMGB1-Tg mice compared with WT mice. Nuclear HMGB1 expression was preserved in HMGB1-Tg mice compared with WT mice and significantly attenuated DNA damage after TAC was attenuated in HMGB1-TG mice.ConclusionThese results suggest that the maintenance of stable nuclear HMGB1 levels prevents hypertrophy and heart failure by inhibiting DNA damage.

MiR-30a downregulation aggravates pressure overload-induced cardiomyocyte hypertrophy

miRNAs play an important role in the pathogenesis of cardiac hypertrophy and dysfunction. However, little is known about how miR-30a regulates cardiomyocyte hypertrophy. In the study, Male C57BL/6 mice were subjected to thoracic aortic constriction, and hearts were harvested at 3 weeks. We assayed miR-30a expression level by real-time PCR and defined the molecular mechanisms of miR-30a-mediated cardiomyocyte hypertrophy. We found that myocardial expression of miR-30a was decreased in mouse models of hypertrophy and in H9c2 cells treated with phenylephrine. MiR-30a inhibition markedly increased mRNA expression of cardiac hypertrophy markers such as atrial natriuretic factor and brain natriuretic peptide in H9c2, and cell size was increased after miR-30a inhibitor treatment. Downregulated miR-30a activated autophagy by inhibiting beclin-1 expression in H9c2 cell. More important, autophagy inhibition suppressed miR-30a inhibitor-induced cardiomyocyte hypertrophy. Together, our data demonstrated that downregulated miR-30a aggravates pressure overload-induced cardiomyocyte hypertrophy by activating autophagy, thus offering a new target for the therapy of cardiomyocyte hypertrophy.

MiR-378 Controls Cardiac Hypertrophy by Combined Repression of Mitogen-Activated Protein Kinase Pathway Factors

Several microRNAs (miRs) have been shown to regulate gene expression in the heart, and dysregulation of their expression has been linked to cardiac disease. miR-378 is strongly expressed in the mammalian heart but so far has been studied predominantly in cancer, in which it regulates cell survival and tumor growth. Here, we report tight control of cardiomyocyte hypertrophy through miR-378. In isolated primary cardiomyocytes, miR-378 was found to be both necessary and sufficient to repress cardiomyocyte hypertrophy. Bioinformatic prediction suggested that factors of the mitogen-activated protein kinase (MAPK) pathway are enriched among miR-378 targets. Using mRNA and protein expression analysis along with luciferase assays, we validated 4 key components of the MAPK pathway as targets of miR-378: MAPK1 itself, insulin-like growth factor receptor 1, growth factor receptor-bound protein 2, and kinase suppressor of ras 1. RNA interference with these targets prevented the prohypertrophic effect of antimiR-378, suggesting their functional relation with miR-378. Because miR-378 significantly decreases in cardiac disease, we sought to compensate for its loss through adeno-associated virus-mediated, cardiomyocyte-targeted expression of miR-378 in an in vivo model of cardiac hypertrophy (pressure overload by thoracic aortic constriction). Restoration of miR-378 levels significantly attenuated thoracic aortic constriction-induced cardiac hypertrophy and improved cardiac function. Our data identify miR-378 as a regulator of cardiomyocyte hypertrophy, which exerts its activity by suppressing the MAPK signaling pathway on several distinct levels. Restoration of disease-associated loss of miR-378 through cardiomyocyte-targeted adeno-associated virus-miR-378 may prove to be an effective therapeutic strategy in myocardial disease.

Role of the Calcium-Sensing Receptor in Cardiomyocyte Apoptosis via the Sarcoplasmic Reticulum and Mitochondrial Death Pathway in Cardiac Hypertrophy and Heart Failure

Aims: Alterations in calcium homeostasis in the intracellular endo/sarcoplasmic reticulum (ER/SR) and mitochondria of cardiomyocytes cause cell death via the SR and mitochondrial apoptotic pathway, contributing to ventricular dysfunction. However, the role of the calcium-sensing receptor (CaR) in cardiac hypertrophy and heart failure has not been studied. This study examined the possible involvement of CaR in the SR and mitochondrial apoptotic pathway in an experimental model of heart failure. Methods and Results: In Wistar rats, cardiac hypertrophy and heart failure were induced by subcutaneous injection of isoproterenol (Iso). Calindol, an activator of CaR, and calhex231, an inhibitor of CaR, were administered by caudal vein injection. Cardiac remodeling and left ventricular function were then analyzed in these rats. After 2, 4, 6 and 8 weeks after the administration of Iso, the rats developed cardiac hypertrophy and failure. The cardiac expression of ER chaperones and related apoptotic proteins was significantly increased in the failing hearts. Furthermore, the expression of ER chaperones and the apoptotic rate were also increased with the administration of calindol, whereas the expression of these proteins was reduced with the treatment of calhex231. We also induced cardiac hypertrophy and failure via thoracic aorta constriction (TAC) in mice. After 2 and 4 weeks of TAC, the expression of ER chaperones and apoptotic proteins were increased in the mouse hearts. Furthermore, Iso induced ER stress and apoptosis in cultured cardiomyocytes, while pretreatment with calhex231 prevented ER stress and protected the myocytes against apoptosis. To further investigate the effect of CaR on the concentration of intracellular calcium, the calcium concentration in the SR and mitochondria was determined with Fluo-5N and x-rhod-1 and the mitochondrial membrane potential was examined with JC-1 using laser confocal microscopy. After treatment with Iso for 48 hours, activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_m, decreased the mitochondrial membrane potential, increased the expression of ER stress chaperones and related apoptotic proteins, and induced the release of cytochrome c from the mitochondria. Conclusions: Our results demonstrated that CaR activation caused Ca²⁺ release from the SR into the mitochondria and induced cardiomyocyte apoptosis through the SR and mitochondrial apoptotic pathway in failing hearts.

Cardiomyocyte Ca2+ handling and structure is regulated by degree and duration of mechanical load variation

Cardiac transverse (t)-tubules are altered during disease and may be regulated by stretch-sensitive molecules. The relationship between variations in the degree and duration of load and t-tubule structure remains unknown, as well as its implications for local Ca2+-induced Ca2+ release (CICR). Rat hearts were studied after 4 or 8 weeks of moderate mechanical unloading [using heterotopic abdominal heart–lung transplantation (HAHLT)] and 6 or 10 weeks of pressure overloading using thoracic aortic constriction. CICR, cell and t-tubule structure were assessed using confocal-microscopy, patch-clamping and scanning ion conductance microscopy. Moderate unloading was compared with severe unloading [using heart-only transplantation (HAHT)]. Mechanical unloading reduced cardiomyocyte volume in a time-dependent manner. Ca2+ release synchronicity was reduced at 8 weeks moderate unloading only. Ca2+ sparks increased in frequency and duration at 8 weeks of moderate unloading, which also induced t-tubule disorganization. Overloading increased cardiomyocyte volume and disrupted t-tubule morphology at 10 weeks but not 6 weeks. Moderate mechanical unloading for 4 weeks had milder effects compared with severe mechanical unloading (37% reduction in cell volume at 4 weeks compared to 56% reduction after severe mechanical unloading) and did not cause depression and delay of the Ca2+ transient, increased Ca2+ spark frequency or impaired t-tubule and cell surface structure. These data suggest that variations in chronic mechanical load influence local CICR and t-tubule structure in a time- and degree-dependent manner, and that physiological states of increased and reduced cell size, without pathological changes are possible.

INVESTIGATION OF MECHANISMS UNDERLYING THE INTERACTION BETWEEN NOX2 NADPH OXIDASE AND PPAR-α IN LEFT VENTRICULAR HYPERTROPHY

NADPH oxidases and peroxisome proliferator–activated receptor-α (PPAR-α) play key roles in left ventricular hypertrophy (LVH) with emerging evidence supporting an important interaction. To investigate the nature of this interplay, gene-modified...

Inhibition of IκB phosphorylation prevents load-induced cardiac dysfunction in mice

Pressure overload is known to be a cause of cardiac hypertrophy that often transits to heart failure. Although nuclear factor (NF)-κB is a key factor in the progression of cardiac hypertrophy, its pathophysiology is yet to be elucidated. Thus, we aimed to show that inhibition of NF-κB activation improves pressure overload-induced cardiac dysfunction. To assess the effect of inhibition on NF-κB activation in pressure overload cardiac hypertrophy, we used IMD-1041 in a murine thoracic aortic constriction (TAC) model. IMD-1041 inhibits the phosphorylation of IκB via inhibition of IκB kinase-β. IMD-1041 (100 mg·kg(-1)·day(-1)) or vehicle was administered orally into mice once a day, and mice were euthanized on day 42 after TAC. TAC resulted in left ventricular wall thickening, cardiac dysfunction, and increases of heart and lung weight, whereas IMD-1041 significantly suppressed the development of cardiac hypertropy 6 wk after TAC. Histologically, developed cardiac fibrosis and cardiomyocyte hypertrophy occurred in the vehicle-treated group, whereas IMD-1041 significantly attenuated these changes. IMD-1041 suppressed the expression of p65-positive cells and nuclear translocation of p65 induced by TAC compared with vehicle. Matrix metalloproteinase-2 activity increased in the vehicle + TAC-treated group; however, it was suppressed in the IMD-1041 + TAC-treated group. IMD-1041 treatment from day 28 to day 42 after TAC significantly attenuated the decrease in the percentage of fractional shortening and cardiac fibrosis without an antihypertrophic effect. In conclusion, IMD-1041 may be useful for preventing pressure overload-induced cardiac dysfunction and the transition of cardiac hypertrophy to contraction failure via suppression of NF-κB activation.

Abstract 28: In Vivo Knockdown of Mirna-15b Results in Increased Cardiac Hypertrophy and Fibrosis in Response to Pressure Overload of the Mouse Heart

MiRNAs play an important role in the control of diverse aspects of cardiac function. MiR-15b is highly expressed in the heart and is found consistently upregulated in hypertrophic and failing hearts. To investigate the function of miR-15b in the heart we set out two experiments. In the first experiment we generated two independent transgenic mouse lines that drive miR-15b expression under the αMHC-promotor and show a three and four fold overexpression of miR-15b. Strikingly, both lines show a decrease in heart weight/tibia length of 20% in adult and aged mice when compared to littermate controls. We investigated the response of these transgenic mice to thoracic aorta constriction (TAC) and found no differences in the hypertrophic response or in cardiac function measured by echocardiography between wild-type and transgenic mice. In a second experiment, we inhibited miR-15b using LNA-based antimiRs. In these mice, TAC resulted in an increased hypertrophic response and increased cardiac fibrosis when compared to a negative control antimiR. A wide range of predicted targets of miR-15 belong to the pathways of the TGFβ-superfamily and using a smad-dependent reporter we show that miR-15b inhibits TGFβ-induced Smad activity in HepG2 cells. One of the predicted targets in the TGFβ pathway is TGFβ receptor 1 (TGFβR1), of which the 3’UTR contains six predicted miR-15 binding sites. This suggests that the phenotype in the transgenic mice and after knockdown of miR-15b may be (partly) mediated by repression of TGFβR1. Indeed, in the adult miR-15b transgenic hearts we found a downregulation of TGFβR1 mRNA and protein and we confirmed binding of miR-15 to the TGFβR1 3’UTR by luciferase assays. In conclusion, miR-15b causes a cardiac hypotrophic phenotype at baseline in transgenic mice and inhibition of miR-15b leads to a stronger hypertrophic and fibrotic response after TAC. Furthermore miR-15b inhibits the TGFβ pathway by targeting the TGFβR1 and possibly other targets in this pathway. This research is funded by the Dutch Heart Foundation (NHF grant #2007B077).

Unbiased plasma proteomics for novel diagnostic biomarkers in cardiovascular disease: identification of quiescin Q6 as a candidate biomarker of acutely decompensated heart failure

Biochemical marker testing has improved the evaluation and management of patients with cardiovascular diseases over the past decade. Natriuretic peptides (NPs), used in clinical practice to assess cardiac dysfunction, exhibit many limitations, however. We used an unbiased proteomics approach for the discovery of novel diagnostic plasma biomarkers of heart failure (HF). A proteomics pipeline adapted for very low-abundant plasma proteins was applied to clinical samples from patients admitted with acute decompensated HF (ADHF). Quiescin Q6 (QSOX1), a protein involved in the formation of disulfide bridges, emerged as the best performing marker for ADHF (with an area under the receiver operator characteristic curve of 0.86, 95% confidence interval: 0.79-0.92), and novel isoforms of NPs were also identified. Diagnostic performance of QSOX1 for ADHF was confirmed in 267 prospectively collected subjects of whom 76 had ADHF. Combining QSOX1 to B-type NP (BNP) significantly improved diagnostic accuracy for ADHF by particularly improving specificity. Using thoracic aortic constriction in rats, QSOX1 was specifically induced within both left atria and ventricles at the time of HF onset. The novel biomarker QSOX1 accurately identifies ADHF, particularly when combined with BNP. Through both clinical and experimental studies we provide lines of evidence for a link between ADHF and cardiovascular production of QSOX1.

Mechanisms Protecting Chronic Pressure Overload by Apoptosis Inhibition

It is generally believed that chronic pressure overload increases myocyte apoptosis, and that the resultant loss of myocytes is responsible for the cardiac dysfunction. If this reasoning is correct, then protecting myocyte apoptosis with chronic pressure overload should increase numbers of myocytes. We examined the effects of protecting apoptosis by caspase inhibition (CI) with chronic pressure overload induced by thoracic aortic constriction (TAC). Left ventricular (LV) ejection fraction was reduced by TAC (55% vs 70%), but was protected with CI (66%). However, CI reduced myocyte apoptosis by only 0.06%, compared with non‐myocyte apoptosis (0.21%), and did not result in increased total myocyte number, which was similar in sham operated controls to TAC with and without CI. However, other mechanisms were potentially responsible for the protection of cardiac function: 1) CI reduced interstitial fibrosis (2.9%) compared to vehicle (6.8%) after TAC. 2) Ki67 staining of endothelial cells was increased with CI and TAC (3.4±0.7%) compared to vehicle group (1.4±0.6%) indicative of angiogenesis. 3) Ki67‐positive myocytes, indicative of myogenesis, were also higher with CI than vehicle (0.9±0.2% vs 0.3±0.1%). Thus, CI improved LV function after TAC, not by protecting myocyte apoptosis, but rather through angiogenesis and myogenesis, reducing myocardial fibrosis.

In vivo cardiac 31P MRS in a mouse model of heart failure

ObjectiveTo investigate myocardial energy status in a mouse model of heart failure using in vivo 31P magnetic resonance spectroscopy (MRS).MethodsMale C57BL/6 mice underwent thoracic aortic constriction (TAC) surgery, inducing pressure‐overload cardiomyopathy, and were measured seven weeks after surgery (n = 5). Healthy wild‐type mice served as controls (n = 4). Cardiac cine 1H MR images were made for reference purposes and to quantify left ventricular (LV) function. Cardiac 31P MR spectra were measured from a ~6 mm cubic voxel enclosing the end‐diastolic LV myocardium using ECG triggered, respiratory gated 3D Image‐Selected In vivo Spectroscopy (ISIS).ResultsLV end‐diastolic volume and LV mass normalized to body weight were higher in TAC mice compared to controls (91.7 ± 19.0 versus 61.8 ± 6.0 μL and 4.4 ± 0.6 versus 3.1 ± 0.2 mg/g, P &lt; 0.01), whereas LV ejection fraction was reduced in TAC mice (45.4 ± 20.0 versus 64.4 ± 5.2 %, P &lt; 0.05). Myocardial phosphocreatine‐to‐ATP ratio was lower in TAC mice when compared to healthy controls (0.8±0.2 versus 1.2±0.2, P &lt; 0.05).ConclusionDecreased EF in TAC mice is accompanied by decreased phosphocreatine‐to‐ATP ratio, indicating a disturbed energy homeostasis in this mouse model of heart failure. This research was funded by a VIDI grant from the Netherlands Organisation for Scientific Research (NWO).

T-Tubule Morphology and Local Ca2+-Induced Ca2+ Release in Rat Ventricular Cardiomyocytes are Sensitive to Chronic Changes in Mechanical Load of the Heart

Transverse (t) tubules optimise cardiomyocyte excitation-contraction coupling by promoting cross-talk between L-type Ca2+ channels and ryanodine receptors, and are disrupted in several cardiac diseases, leading to heart failure. A unifying hypothesis is that t-tubule structure is altered by chronically varying the mechanical load of the heart, consequently disrupting local Ca2+-induced Ca2+ release (CICR).To test this hypothesis, rat hearts were unloaded for 4 or 8 weeks using heterotopic abdominal heart-lung transplantation, or overloaded for 6 or 10 weeks using thoracic aortic constriction. Left ventricular cardiomyocytes were isolated enzymatically and studied using confocal microscopy.Chronic overloading increased cardiomyocyte volume at 10 but not 6 weeks (Control 45,348±2373μm3, n=47 vs. OV 6 weeks 48,785±2237μm3, n=60 vs. OV 10 weeks 56,066±3091μm3, n=42; p<0.05) and disrupted t-tubule regularity, which was measured as the power of the dominant frequency of the Fourier transform of Di-8-ANEPPS images (Control 1.69±0.1x107, n=44 vs. OV 10 weeks 9.45±1.0x106, n=33; p<0.001), despite preserved Ca2+ transients. Unloading decreased cardiomyocyte size and induced time-dependent Ca2+ transient changes. Ca2+ release synchronicity (measured by the variance of the time-to-peak of the Ca2+ transient) was disrupted at 8 but not 4 weeks (Control 269.6±17.13ms2, n=106 vs. UN 4 weeks 331.6±23.67ms2, n=46 vs. UN 8 weeks 403.5±35.4ms2, n=50; p<0.01), as was the t-tubule regularity (Control 1.59±0.01x107, n=107 vs. UN 4 weeks 1.75±0.1x107, n=53 vs. UN 8 weeks 1.1±0.0007x107, n=82; p<0.01).We demonstrate that an increase or decrease in load results in time-dependent disrupted t-tubule morphology. This can provide a mechanism for inefficient CICR observed in chronic heart failure and the functional deterioration after prolonged unloading of the heart using left ventricular assist devices.