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Many biomarkers that could be used to assess ejection fraction, heart failure, or myocardial infarction fail to translate into clinical practice because they lack essential performance characteristics or fail to meet regulatory standards for approval. Despite their potential, new technologies have added to the complexities of successful translation into clinical practice. Biomarker discovery and implementation require a standardized approach that includes: identification of a clinical need; identification of a valid surrogate biomarker; stepwise assay refinement, demonstration of superiority over current standard-of-care; development and understanding of a clinical pathway; and demonstration of real-world performance. Successful biomarkers should improve efficacy or safety of treatment, while being practical at a realistic cost. Everyone involved in cardiovascular healthcare, including researchers, clinicians, and industry partners, are important stakeholders in facilitating the development and implementation of biomarkers. This article provides suggestions for a development pathway for new biomarkers, discusses regulatory issues and challenges, and suggestions for accelerating the pathway to improve patient outcomes. Real-life examples of successful biomarkers—high-sensitivity cardiac troponin, T2* cardiovascular magnetic resonance imaging, and echocardiography—are used to illustrate the value of a standardized development pathway in the translation of concepts into routine clinical practice. [ABSTRACT FROM AUTHOR]

Aims Chronic heart failure is becoming increasingly prevalent and is still associated with a high mortality rate. Myocardial hypertrophy and fibrosis drive cardiac remodelling and heart failure, but they are not sufficiently inhibited by current treatment strategies. Furthermore, despite increasing knowledge on cardiomyocyte intracellular signalling proteins inducing pathological hypertrophy, therapeutic approaches to target these molecules are currently unavailable. In this study, we aimed to establish and test a therapeutic tool to counteract the 22 kDa calcium and integrin binding protein (CIB) 1, which we have previously identified as nodal regulator of pathological cardiac hypertrophy and as activator of the maladaptive calcineurin/NFAT axis. Methods and results Among three different sequences, we selected a shRNA construct (shCIB1) to specifically down-regulate CIB1 by 50% upon adenoviral overexpression in neonatal rat cardiomyocytes (NRCM), and upon overexpression by an adeno-associated-virus (AAV) 9 vector in mouse hearts. Overexpression of shCIB1 in NRCM markedly reduced cellular growth, improved contractility of bioartificial cardiac tissue and reduced calcineurin/NFAT activation in response to hypertrophic stimulation. In mice, administration of AAV-shCIB1 strongly ameliorated eccentric cardiac hypertrophy and cardiac dysfunction during 2 weeks of pressure overload by transverse aortic constriction (TAC). Ultrastructural and molecular analyses revealed markedly reduced myocardial fibrosis, inhibition of hypertrophy associated gene expression and calcineurin/NFAT as well as ERK MAP kinase activation after TAC in AAV-shCIB1 vs. AAV-shControl treated mice. During long-term exposure to pressure overload for 10 weeks, AAV-shCIB1 treatment maintained its anti-hypertrophic and anti-fibrotic effects, but cardiac function was no longer improved vs. AAV-shControl treatment, most likely resulting from a reduction in myocardial angiogenesis upon downregulation of CIB1. Conclusions Inhibition of CIB1 by a shRNA-mediated gene therapy potently inhibits pathological cardiac hypertrophy and fibrosis during pressure overload. While cardiac function is initially improved by shCIB1, this cannot be kept up during persisting overload. [ABSTRACT FROM AUTHOR]

Aims: Pulmonary hypertension is a progressive disease with poor prognosis, characterized by pathological inward remodelling and loss of patency of the lung vasculature. The right ventricle is co-affected by pulmonary hypertension, which triggers events such as hypoxia and/or increased mechanical load. Initially the right ventricle responds with 'adaptive' hypertrophy, which is often rapidly followed by 'maladaptive' changes leading to right heart decompensation and failure, which is the ultimate cause of death. Methods and results: We report here that miR-223 is expressed in the murine lung and right ventricle at higher levels than in the left ventricle. Moreover, lung and right-ventricular miR-223 levels were markedly down-regulated by hypoxia. Correspondingly, increasing right-ventricular load by pulmonary artery banding, induced right-ventricular ischaemia, and the downregulation of miR-223. Lung and right ventricle miR-223 down-regulation were linked with increased expression of the miR-223 target; insulin-like growth factor-I receptor (IGF-IR) and IGF-I downstream signalling. Similarly, miR-223 was decreased and IGF-IR increased in human pulmonary hypertension. Notably in young mice, miR-223 overexpression, the genetic inactivation or pharmacological inhibition of IGF-IR, all attenuated right-ventricular hypertrophy and improved right heart function under conditions of hypoxia or increased afterload. Conclusion: These findings highlight the early role of pulmonary and right-ventricular miR-223 and the IGF-IR in the right heart failure programme initiated by pulmonary hypoxia and increased mechanical load and may lead to the development of novel therapeutic strategies that target the development of PH and right heart failure. [ABSTRACT FROM AUTHOR]

Abstract: With evolving knowledge in molecular and cellular cardiology, cardiac gene therapy has already been investigated in clinical studies. Different vector systems for cardiac gene therapy have been developed in recent years. While non-viral vectors, such as plasmid DNA, allow remarkable organ specificity, they are often limited by low transfection efficiency and transient gene expression. In contrast, adenoviral or adeno-associated virus-based vectors transfer the transgene more efficiently, but organ specificity may be reduced and immunogenic properties can limit their applicability. Using advanced transcriptional and transductional targeting strategies, viral vectors have been improved in the last few years. Recently, more efficient serotypes of adeno-associated viruses have been identified that show increased transduction rates, thus reducing the necessity for high virus titers. Combination with specific application techniques, such as intramyocardial injection, catheter-based perfusion, ultrasound targeted microbubble destruction, or retroinfusion may further enhance vector efficiency. This review article will give a broad overview of different gene delivery strategies that have been applied in experimental and clinical studies targeting the heart. [Copyright &y& Elsevier]

Abstract: Objective: Vectors based on recombinant adeno-associated virus 2 (AAV-2) are a promising tool for cardiac gene transfer. However, potential therapeutic applications need to consider the predominant transduction of the liver once AAV-2 vectors enter the systemic circulation. We therefore aimed to increase efficiency and specificity of cardiac vector delivery by combining transcriptional and cell surface targeting. Methods: For analysis of transcriptional targeting, recombinant AAV vectors were generated harboring a luciferase reporter gene under control of the cytomegalovirus (CMV) promoter or the 1.5-kb cardiac myosin light chain promoter fused to the CMV immediate-early enhancer (CMVenh/MLC1.5). Luciferase activities were determined in representative organs three weeks after intravenous injection of the vector into adult mice. Transductional targeting was studied using luciferase-reporter constructs crosspackaged into capsids of AAV serotypes 1 to 6 and modified AAV-2 capsids devoid of binding their primary receptor heparan sulfate proteoglycan. Results: Intravenous injections of AAV-2 vectors harboring the CMVenh/MLC1.5 promoter enabled a specific and 50-fold higher reporter gene expression in left ventricular myocardium of adult mice compared to vectors containing the CMV promoter. Comparison of AAV-2 vector genomes crosspackaged into capsids of AAV-1 to -6 showed that AAV-1, -4, -5, and -6 capsids increased cardiac transduction efficiency by about 10-fold. However, transduction of other organs such as the liver was also increased after systemic administration. In contrast, AAV-2-based vectors with ablated binding to their primary receptor heparan sulfate proteoglycan enabled a significantly increased efficiency of cardiac gene transfer and reduced transduction of the liver. Conclusions: Combining transcriptional targeting by the CMVenh/MLC1.5 promoter and AAV vectors devoid of binding the AAV-2 primary receptor results in an efficient cardiac gene transfer with a significantly reduced hepatic transduction. [Copyright &y& Elsevier]

Abstract: Objective: Hereditary long QT syndrome (LQTS) is a genetically heterogeneous disease characterized by prolonged QT intervals and an increased risk for ventricular arrhythmias and sudden cardiac death. Mutations in the voltage-gated potassium channel subunit KCNQ1 induce the most common form of LQTS. KCNQ1 is associated with two different entities of LQTS, the autosomal-dominant Romano–Ward syndrome (RWS), and the autosomal-recessive Jervell and Lange–Nielsen syndrome (JLNS) characterized by bilateral deafness in addition to cardiac arrhythmias. In this study, we investigate and discuss dominant-negative I Ks current reduction by a KCNQ1 deletion mutation identified in a RWS family. Methods: Single-strand conformation polymorphism analysis and direct sequencing were used to screen LQTS genes for mutations. Mutant KCNQ1 channels were heterologously expressed in Xenopus oocytes, and potassium currents were recorded using the two-microelectrode voltage clamp technique. Results: A heterozygous deletion of three nucleotides (CTT) identified in the KCNQ1 gene caused the loss of a single phenylalanine residue at position 339 (KCNQ1-ΔF339). Electrophysiological measurements in the presence and absence of the regulatory β-subunit KCNE1 revealed that mutant and wild type forms of an N-terminal truncated KCNQ1 subunit (isoform 2) caused much stronger dominant-negative current reduction than the mutant form of the full-length KCNQ1 subunit (isoform 1). Conclusion: This study highlights the functional relevance of the truncated KCNQ1 splice variant (isoform 2) in establishment and mode of inheritance in long QT syndrome. In the RWS family presented here, the autosomal-dominant trait is caused by multiple dominant-negative effects provoked by heteromultimeric channels formed by wild type and mutant KCNQ1-isoforms in combination with KCNE1. [Copyright &y& Elsevier]

Abstract: Objective: The human ether-a-go-go-related gene (hERG) encodes the rapid component of the cardiac repolarizing delayed rectifier potassium current, IKr. The direct interaction of the commonly used protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM I) with hERG, KvLQT1/minK, and IKr currents was investigated in this study. Methods: hERG and KvLQT1/minK channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. In addition, hERG currents in stably transfected human embryonic kidney (HEK 293) cells, native IKr currents and action potentials in isolated guinea pig ventricular cardiomyocytes were recorded using whole-cell patch clamp electrophysiology. Results: Bisindolylmaleimide I blocked hERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner with IC50 values of 1.0 and 13.2 μM, respectively. hERG channels were primarily blocked in the open state in a frequency-independent manner. Analysis of the voltage-dependence of block revealed a reduction of inhibition at positive membrane potentials. BIM I caused a shift of −20.3 mV in the voltage-dependence of inactivation. The point mutations tyrosine 652 alanine (Y652A) and phenylalanine 656 alanine (F656A) attenuated hERG current blockade, indicating that BIM I binds to a common drug receptor within the pore region. KvLQT1/minK currents were not significantly altered by BIM I. Finally, 1 μM BIM I reduced native IKr currents by 69.2% and lead to action potential prolongation. Conclusion: In summary, PKC-independent effects have to be carefully considered when using BIM I as PKC inhibitor in experimental models involving hERG channels and IKr currents. [Copyright &y& Elsevier]

Objective: The cardiac inwardly rectifying potassium current IK1 and its molecular correlates Kir2.1 and Kir2.2 play an important role in cardiac repolarisation and in the pathogenesis of hereditary long-QT syndrome (LQTS-7). Protein kinases A (PKA) and C (PKC) are key enzymes in adrenergic signal transduction, inducing arrhythmias in heart disease. This study investigated the regulation of Kir2.2 (KCNJ12) by PKA. Methods: Cloned Kir2.2 channels were expressed heterologously in Xenopus oocytes and currents were measured with the double-electrode voltage–clamp technique. Results: After activation of PKA by forskolin (100 μmol/l) or Ro-20-1724 (100 μmol/l), wild type currents at -120 mV were increased by 93.7% and 79.0%, respectively. Coapplication of the PKA inhibitor KT-5720 (2.5 μmol/l) attenuated this effect. No significant changes were apparent after mutation of the single PKA consensus site S430. In addition, removal of all four PKC consensus sites in Kir2.2 induced a phorbolester-mediated current increase which could be suppressed by PKA inhibitors H-89 (50 μmol/l) and KT-5720 (2.5 μmol/l). Conclusions: This study demonstrates antagonistic effects of PKA and PKC in the regulation of Kir2.2. Phosphorylation by PKC has been shown to cause an inhibition of Kir2.2 currents, whereas activation of PKA leads to current upregulation. [Copyright &y& Elsevier]

Human hereditary long QT syndrome is a cardiac disease characterized by prolongation of the QT interval and increased susceptibility to ventricular arrhythmias and sudden cardiac death. Mutations in the human-ether-a-go-go-related gene (hERG), encoding the protein underlying the repolarizing cardiac IKr potassium current, cause chromosome 7-linked long QT syndrome 2. Loss of function of mutant hERG channels may be caused by several mechanisms, including altered current kinetics, altered ion selectivity, or defective intracellular protein trafficking. Especially the latter category has become a focus of particular interest recently, because some of the mutant subunits display wild type current properties when normal trafficking is restored and channels are inserted in the cell membrane in vitro. This review summarizes the current knowledge on hERG channel trafficking under physiological and pathological conditions. In addition, therapeutic approaches to restore normal hERG trafficking in vitro and in vivo are discussed. [Copyright &y& Elsevier]

Objective: The activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA) is reduced in the failing myocardium. Therefore, transfer of SERCA2a cDNA is considered as a therapeutical approach. The aim of this study was analysis of the long-term effect of SERCA2a overexpression in normal as well as pressure overload challenged myocardium of transgenic rats. Methods: Independent transgenic rat lines were established expressing the rat SERCA2a cDNA specifically in the myocardium resulting in increased SERCA2a protein levels by 30–70%. Simultaneous measurements of isometric contraction and calcium transients were carried out in right ventricular papillary muscle preparations. Hemodynamic parameters were measured in hearts of unchallenged rats as well as 10 weeks after pressure overload induced by abdominal aortic banding. Results: Analysis of calcium handling and contractile parameters in isolated right ventricular papillary muscles revealed significant shortening of intracellular calcium transients and half maximal relaxation times (RT50). Assessing myocardial contractility in working heart preparations, both transgenic rat lines revealed elevated left ventricular pressure, improved systolic and diastolic parameters, attenuated negative force–frequency relation, and a dose-dependent β-adrenergic effect. Aortic banding resulted in reduction of left ventricular pressure and worsening of contraction and relaxation parameters with no differences in mortality in both transgenic (+dP/dt 3084±96 vs. 3938±250 mmHg/s; RT50 47.0±1.2 vs. 36.7±1.4 ms) and wild-type rats (+dP/dt 2695±86 vs. 3297±122 mmHg/s; RT50 53.0±1.6 vs. 44.1±1.4). SERCA2a overexpressing hearts revealed improved hemodynamic parameters compared to wild-type controls. Acceleration of isovolumetric relaxation characterized by the index Tau was directly correlated to SERCA2a protein concentrations. Conclusion: Overexpression of SERCA2a protein results in a positive inotropic effect under baseline conditions remaining preserved under pressure overload without affecting mortality. Therefore therapeutic transfer of SERCA2a may become a potential approach for gene therapy of congestive heart failure. Moreover, transgenic SERCA2a rats will be useful for studies of long-term SERCA2a overexpression in further cardiovascular disease models. [Copyright &y& Elsevier]

Objective: Patients with HERG-associated long QT syndrome typically develop tachyarrhythmias during physical or emotional stress. Previous studies have revealed that activation of the beta-adrenergic system and consecutive elevation of the intracellular cAMP concentration regulate HERG channels via protein kinase A-mediated phosphorylation of the channel protein and via direct interaction with the cAMP binding site of HERG. In contrast, the influence of the alpha-adrenergic signal transduction cascade on HERG currents as suggested by recent reports is less well understood. The aim of the present study was to elucidate the biochemical pathways of the protein kinase C (PKC)-dependent regulation of HERG currents. Methods: HERG channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. Results: Application of the phorbol ester PMA, an unspecific protein kinase activator, shifted the voltage dependence of HERG activation towards more positive potentials. This effect could be mimicked by activation of conventional PKC isoforms with thymeleatoxin. Coexpression of HERG with the beta-subunits minK or hMiRP1 did not alter the effect of PMA. Specific inhibition of PKC abolished the PMA-induced activation shift, suggesting that PKC is required within the regulatory mechanism. The PMA-induced effect could still be observed when the PKC-dependent phosphorylation sites in HERG were deleted by mutagenesis. Cytoskeletal proteins such as actin filaments or microtubules did not affect the HERG activation shift. Conclusion: In addition to the known effects of PKA and cAMP, HERG channels are also modulated by PKC. The molecular mechanisms of this PKC-dependent process are not completely understood but do not depend on direct PKC-dependent phosphorylation of the channel. [Copyright &y& Elsevier]

Objective: Blood levels of cytokines are commonly elevated in severe congestive heart failure (CHF) and in coronary artery disease (CAD). While the adverse effects of cytokines on contractile function and myocardial cell integrity are well studied, little is known on whether cardiac cells are only targets or active players in these inflammatory reactions. Methods and results: We tested if human coronary artery endothelial cells (HCAEC) may become a source of cytokine and adhesion molecule expression when stimulated with bacterial lipopolysaccharide (LPS). Analysis of HCAEC supernatants by ELISA identified enhanced secretion of IL-6, IL-8, and MCP-1 while endothelin-1 was not increased. IL-1β, IL-10, or TNF-α were not detectable by ELISA while RT-PCR revealed enhanced mRNA expression of IL-1β and TNF-α but not IL-10. FACS analysis showed an LPS-induced upregulation of ICAM-1, VCAM, and ELAM-1. LFA-1 could not be detected. We further characterized receptors involved in LPS-induced signaling. Our results indicate that activation of HCAEC by LPS requires Toll-like receptor (TLR) 4. Pretreating the cells with the 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase inhibitor Cerivastatin reduced IL-6 release. Conclusions: Taken together, our results indicate that activated HCAEC may act as inflammatory cells and thus directly contribute to the progression of CHF and CAD. [Copyright &y& Elsevier]

Objective: Adrenergic stimulation of the heart leads to activation of the phospholipase D signal transduction pathway with formation of the intracellular second messengers phosphatidic acid and diacylglycerol, which may play a role in the development of myocardial hypertrophy by activating mitogen-activated protein kinases and protein kinase C. So far, the adrenergic receptor subtypes mediating activation of cardiac phospholipase D are not known. Methods: We developed an assay for determination of phospholipase D activity in the isolated perfused rat heart. Utilizing the phospholipase D specific transphosphatidylation reaction the stable product phosphatidylethanol (PEtOH) is formed in rat hearts perfused in the presence of 1% ethanol. Myocardial PEtOH formation was used as a marker of phospholipase D activity and was determined by HPLC and evaporative light-scattering detection (PEtOH μg/mg myocardial protein). Results: Basal PEtOH formation in unstimulated hearts was 0.06±0.01 μg/mg. Stimulation of the hearts with norepinephrine resulted in a concentration-dependent phospholipase D activation with a maximum formation of PEtOH (0.17±0.01 μg/mg) at 100 μmol/l norepinephrine. The norepinephrine-induced increase in PLD activity was completely blocked by the α1-adrenoceptor antagonist prazosin and was unaffected by the β-adrenoceptor antagonist propranolol. Further characterisation of α1-adrenoceptor subtypes with selective α1-adrenoceptor antagonists demonstrated a complete inhibition of the norepinephrine-induced phospholipase D activation by WB 4101 (α1A-selective: 0.06±0.01 μg/mg) and by BMY 7378 (α1D-selective: 0.07±0.01 μg/mg). In contrast, the α1B-adrenoceptor antagonist chloroethylclonidine had no inhibitory effect on norepinephrine-stimulated phospholipase D activity (0.14±0.01 μg/mg). Conclusion: Adrenergic activation of the cardiac phospholipase D signal transduction pathway is mediated by α1-adrenoceptors. Here, the α1A-adrenoceptor subtype, but not the α1B-adrenoceptor are coupled to activation of cardiac phospholipase D. [Copyright &y& Elsevier]

Objective: Numerous studies support the concept of impaired postischemic sympathetic neurotransmission in the heart. We hypothesized that postischemic neuronal dysfunction (neuronal stunning) is caused by a transient suppression of exocytotic norepinephrine (NE) release from sympathetic nerve terminals. Furthermore, we assessed the role of presynaptic adenosine-receptors and α2-adrenoceptors in neuronal stunning. Methods and results: Exocytotic NE release was induced by two electrical field stimulations (S1 and S2) in isolated perfused rat hearts. S1 was performed under baseline conditions and S2 either during or following intervention. Results are expressed as mean S2/S1 ratios±S.E.M. Stepwise increase of global ischemic periods (10, 20, and 30 min) induced a progressive suppression of NE release in the postischemic hearts, which was reversible during reperfusion. Both the degree and duration of NE suppression was dependent on the extent of the preceding ischemic period. Following 10-min ischemia complete recovery of NE release was achieved after 5-min reperfusion (1.07±0.12), whereas 5-min reperfusion did not restore NE release after 30 min (0.36±0.07) of ischemia. The adenosine-receptor antagonists 8-phenyltheophylline (8-PT; non-selective) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; adenosine A1-receptor subtype selective) significantly increased NE release after 30-min ischemia and 5-min reperfusion (0.78±0.06 and 0.64±0.07), while in the same experimental protocol blockade of α2-adrenoceptors by yohimbine failed to restore the postischemic release (0.24±0.06). In non-ischemic hearts the adenosine analogue R(−)N6-(2-phenylisopropyl)adenosine (R-PIA) resulted in a marked suppression of NE release (0.61±0.07). The inhibitory effect of R-PIA and 2-chloro-N6-cyclopentyladenosine (CCPA; adenosine A1-receptor subtype selective agonist) persisted 5 min after cessation of R-PIA (0.62±0.05) and CCPA (0.58±0.04). Activation of α2-adrenoceptors by 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 14,304) also caused a reduction of NE release (0.50±0.02), but the release increased to control levels 5 min after cessation of UK 14,304 (0.90±0.06). Conclusions: The results establish the phenomenon of neuronal stunning in terms of a postischemic suppression of exocytotic NE release and provide evidence that neuronal stunning is mediated by endogenous adenosine through activation of presynaptic adenosine A1-receptors. [ABSTRACT FROM PUBLISHER]

Objective: Familial hypertrophic cardiomyopathy (FHC) due to mutations of cardiac troponin T (cTnT) is associated with a high frequency of sudden death even in the absence of cardiac hypertrophy. To investigate the causal relationship of cTnT mutations and this particular phenotype, we sought to establish a transgenic rat model for the disease. Methods: Transgenic rats were generated expressing human wild-type cTnT or two truncated cTnT molecules (del ex16, del ex15/16), resulting from an intron 15 splice donor site mutation previously observed in FHC patients. Transgenic rat hearts were characterized by histology, immunohistochemistry and in the ‘working heart’. Results: Human wild-type and del ex16 cTnT were stably expressed and incorporated into the sarcomere of transgenic cardiomyocytes. Del ex16 transgenic rats revealed a lower level of expression (4–5%) than human wt cTnT animals (25–40%). In the ‘working heart’ model del ex16 hearts exhibited significant systolic and diastolic dysfunction without cardiac hypertrophy. In contrast, human wt cTnT hearts showed improved contractile performance and moderate myocardial hypertrophy. After 6 months of daily physical exercise one del ex16 rat died suddenly and three out of five del ex16 hearts revealed ventricular tachycardia/fibrillation. No arrhythmia was observed in human wt cTnT expressors. Myofibrillar disarray was present in del ex16 hearts after training but not in human wild-type cTnT rats or non-transgenic controls. Conclusion: A human cTnT deletion overexpressed in transgenic rats exerts a dominant-negative effect and mimics the phenotype of FHC with diastolic dysfunction and arrhythmias. By contrast, human cTnT wild-type animals reveal a gain of function and cardiac hypertrophy without arrhythmias. [ABSTRACT FROM PUBLISHER]

Background: Smooth muscle cells (SMC) are a preferential target for gene therapeutic approaches in atherosclerosis and restenosis. However, the undesirable expression of putative therapeutic genes in tissues other than the vascular wall is a considerable safety limitation for clinical trials, thus requiring the identification of a smooth-muscle-specific promoter sequence. Since the 2.3 kb rabbit Smooth Muscle Myosin Heavy Chain (SMHC) promoter was shown to be transcriptionally active in primary vascular but not visceral or other non-SMC in vitro, this fragment was chosen for in vivo analysis. Methods and Results: Transgenic mice and rabbits were established expressing a luciferase reporter gene under control of the 2.3 kb rabbit SMHC promoter. In contrast to the endogenous expression pattern of the SMHC gene both species revealed light emission predominantly in the arterial system including coronary arteries. Low activities were measured in large veins and the gastrointestinal system. In situ hybridization of murine embryos using a luciferase riboprobe confirmed reporter gene expression in large arteries with no detectable mRNA in the viscera. Unlike adult animals, ectopic luciferase activities were found in ventricular myocardium during murine development ceasing 1 week post partum. Conclusions: In two animal species, the 2.3 kb SMHC promoter appeared to be effective in discriminating between the pathways regulating vascular and visceral smooth muscle gene expression. The vascular-specific expression profile of the 2.3 kb SMHC promoter suggests that the 2.3 kb SMHC promoter contains the regulatory elements necessary for selective gene targeting into vascular SMC of large arteries including coronary arteries in vivo. [ABSTRACT FROM PUBLISHER]

Objective: To approach heart muscle diseases by gene transfer, an adenoviral vector system was intended to be established suitable for gene expression in ventricular and/or atrial myocardium. Methods: Two adenoviral vectors (Ad-mhcLuc, Ad-mlcLuc) were constructed, in which the luciferase reporter gene is under control of either the ventricle-specific myosin light chain-2 (mlc-2v) or the atrial- and ventricular-specific α-myosin heavy chain (α-mhc) promoter. For controls, a recombinant adenovirus without promoter (Ad-Luc) and one with the Rous sarcoma virus (rsv) promoter (Ad-rsvLuc) were generated. A volume of 20 μl containing 2×109 plaque forming units (pfu) of the recombinant adenoviruses Ad-mhcLuc, Ad-mlcLuc, Ad-rsvLuc or Ad-Luc was injected into the cardiac cavity or the quadriceps femoris muscle of neonatal rats. After five days animals were sacrificed and nine different tissues were analyzed for reporter gene expression by detection of light activity relative to mg of tissue. Results: Injections of recombinant adenoviruses into the cardiac cavity of neonatal rats resulted in heart-specific gene expression of Ad-mlcLuc (20 fold of Ad-Luc; 11% of Ad-rsvLuc), whereas Ad-mhcLuc gave mainly luciferase activity in the heart (6.5 fold of Ad-Luc; 3% of Ad-rsvLuc) with additional activity in lung and liver (2–4 fold of Ad-Luc). In the ventricular tissue Ad-mlcLuc revealed a 35-fold higher luciferase activity, whereas Ad-mhcLuc, Ad-rsvLuc and Ad-Luc showed only 2-fold higher luciferase activities compared to the atrium. Viral DNA in atrial and ventricular tissue was detected by PCR at approximately the same abundance independent of the injected type of adenovirus. Direct injection of Ad-mhcLuc and Ad-mlcLuc into the thigh muscle revealed only background luciferase activities. Conclusions: In the adenoviral system only the mlc-2v promoter may fulfil the safety requirements for a myocardial specific gene expression with a high selectivity for the ventricular myocardium, thus providing a promising tool for future gene therapy of cardiomyopathies. [ABSTRACT FROM PUBLISHER]

After the loss of membrane integrity cardiospecific myosin light chains are released as a result of proteolytic degradation of the insoluble myosin pool. Thus, as with cytosolic enzymes, the measurement of the serum concentration changes of myosin light chains may allow a non-invasive estimation of infarct size. A two compartment model was used to describe the serum concentration changes of myosin light chains after a bolus injection into awake beagle dogs. The intial distribution volume was 7.4% of body weight, whereas the second compartment accounted for 4.7% of body weight. The fractional disappearance rate of 0.0092·min−1 resulted in a serum half life of myosin light chains of 75 min. In 30 patients with acute non-reperfused myocardial infarction a close correlation was found between the cumulative appearance of myosin light chains and their maximal serum concentration. In these patients the cumulative appearance of myosin light chains correlated with serum creatine kinase estimates of infarct size, with impairment of left ventricular ejection fraction, and with mortality during hospital admission. Thus the maximal serum concentration and the cumulative appearance of myosin light chains allow a non-invasive estimation of infarct size and may serve as a serological indicator of the patient's prognosis. [ABSTRACT FROM PUBLISHER]

Aims: TASK-1 (K2P3.1) two-pore-domain potassium channels are atrial-specific and significantly up-regulated in atrial fibrillation (AF) patients, contributing to AF-related electrical remodelling. Inhibition of TASK-1 in cardiomyocytes of AF patients was shown to counteract AF-related action potential duration shortening. Doxapram was identified as a potent inhibitor of the TASK-1 channel. In this study, we investigated the antiarrhythmic efficacy of doxapram in a porcine model of AF., Methods and Results: Doxapram successfully cardioverted pigs with artificially induced episodes of AF. We established a porcine model of persistent AF in domestic pigs via intermittent atrial burst stimulation using implanted pacemakers. All pigs underwent catheter-based electrophysiological investigations prior to and after 14 days of doxapram treatment. Pigs in the treatment group received intravenous administration of doxapram once per day. In doxapram-treated AF pigs, the AF burden was significantly reduced. After 14 days of treatment with doxapram, TASK-1 currents were still similar to values of sinus rhythm animals. Doxapram significantly suppressed AF episodes and normalized cellular electrophysiology by inhibition of the TASK-1 channel. Patch-clamp experiments on human atrial cardiomyocytes, isolated from patients with and without AF could reproduce the TASK-1 inhibitory effect of doxapram., Conclusion: Repurposing doxapram might yield a promising new antiarrhythmic drug to treat AF in patients., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Aims: Heart failure is characterized by structural and metabolic cardiac remodelling. The aim of the present study is to expand our understanding of the complex metabolic alterations in the transition from pathological hypertrophy to heart failure and exploit the results from a translational perspective., Methods and Results: Mice were subjected to transverse aortic constriction (TAC) or sham surgery and sacrificed 2 weeks, 4 weeks, or 6 weeks after the procedure. Samples from plasma, liver, skeletal muscle, and heart were collected and analysed using metabolomics. Cardiac samples were also analysed by transcriptional profiling. Progressive alterations of key cardiac metabolic pathways and gene expression patterns indicated impaired mitochondrial function and a metabolic switch during transition to heart failure. Similar to the heart, liver, and skeletal muscle revealed significant metabolic alterations such as depletion of essential fatty acids and glycerolipids in late stages of heart failure. Circulating metabolites, particularly fatty acids, reflected cardiac metabolic defects, and deteriorating heart function. For example, inverse correlation was found between plasma and the heart levels of triacylglycerol (C18:1, C18:2, C18:3), and sphingomyelin (d18:1, C23:0) already at an early stage of heart failure. Interestingly, combining metabolic and transcriptional data from cardiac tissue revealed that decreased carnitine shuttling and transportation preceded mitochondrial dysfunction. We, thus, studied the therapeutic potential of OCTN2 (Organic Cation/Carnitine Transporter 2), an important factor for carnitine transportation. Cardiac overexpression of OCTN2 using an adeno-associated viral vector significantly improved ejection fraction and reduced interstitial fibrosis in mice subjected to TAC., Conclusion: Comprehensive plasma and tissue profiling reveals systemic metabolic alterations in heart failure, which can be used for identification of novel biomarkers and potential therapeutic targets., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

Aims: Delta-sarcoglycan is a member of the dystrophin-associated glycoprotein complex linking the cytoskeleton to the extracellular matrix. Similar to patients with defects in the gene encoding delta-sarcoglycan (Sgcd), knockout mice develop cardiomyopathy and muscular dystrophy. The aim of our study was to develop an approach for preventing cardiomyopathy in Sgcd-deficient mice by cardiac expression of the intact cDNA upon systemic delivery of adeno-associated viral (AAV) vectors., Methods and Results: We packaged the Sgcd cDNA under transcriptional control of a myosin light chain-promoter fused with a cytomegalovirus enhancer into AAV-9 capsids. Vectors carrying either the Sgcd cDNA or an enhanced green fluorescent protein (EGFP) reporter gene were intravenously injected into adult Sgcd knockout mice. After 6 months, immunohistochemistry revealed almost complete reconstitution of the sarcoglycan subcomplex in heart but not skeletal muscle of mice with the Sgcd vector. Furthermore, Sgcd gene transfer resulted in prevention of cardiac fibrosis and significantly increased running distance measured by voluntary wheel running. Left ventricular function remained stable in mice expressing Sgcd while it deteriorated in EGFP controls within 6 months, paralleled by increased expression of brain natriuretic peptide, a molecular marker of heart failure., Conclusion: Our study establishes an approach to specifically treat hereditary cardiomyopathies by targeting gene expression into the myocardium upon systemic application of AAV vectors.

Objective: Hereditary long QT syndrome (LQTS) is a genetically heterogeneous disease characterized by prolonged QT intervals and an increased risk for ventricular arrhythmias and sudden cardiac death. Mutations in the voltage-gated potassium channel subunit KCNQ1 induce the most common form of LQTS. KCNQ1 is associated with two different entities of LQTS, the autosomal-dominant Romano-Ward syndrome (RWS), and the autosomal-recessive Jervell and Lange-Nielsen syndrome (JLNS) characterized by bilateral deafness in addition to cardiac arrhythmias. In this study, we investigate and discuss dominant-negative I(Ks) current reduction by a KCNQ1 deletion mutation identified in a RWS family., Methods: Single-strand conformation polymorphism analysis and direct sequencing were used to screen LQTS genes for mutations. Mutant KCNQ1 channels were heterologously expressed in Xenopus oocytes, and potassium currents were recorded using the two-microelectrode voltage clamp technique., Results: A heterozygous deletion of three nucleotides (CTT) identified in the KCNQ1 gene caused the loss of a single phenylalanine residue at position 339 (KCNQ1-deltaF339). Electrophysiological measurements in the presence and absence of the regulatory beta-subunit KCNE1 revealed that mutant and wild type forms of an N-terminal truncated KCNQ1 subunit (isoform 2) caused much stronger dominant-negative current reduction than the mutant form of the full-length KCNQ1 subunit (isoform 1)., Conclusion: This study highlights the functional relevance of the truncated KCNQ1 splice variant (isoform 2) in establishment and mode of inheritance in long QT syndrome. In the RWS family presented here, the autosomal-dominant trait is caused by multiple dominant-negative effects provoked by heteromultimeric channels formed by wild type and mutant KCNQ1-isoforms in combination with KCNE1.

Objective: The cardiac inwardly rectifying potassium current IK1 and its molecular correlates Kir2.1 and Kir2.2 play an important role in cardiac repolarisation and in the pathogenesis of hereditary long-QT syndrome (LQTS-7). Protein kinases A (PKA) and C (PKC) are key enzymes in adrenergic signal transduction, inducing arrhythmias in heart disease. This study investigated the regulation of Kir2.2 (KCNJ12) by PKA., Methods: Cloned Kir2.2 channels were expressed heterologously in Xenopus oocytes and currents were measured with the double-electrode voltage-clamp technique., Results: After activation of PKA by forskolin (100 micromol/l) or Ro-20-1724 (100 micromol/l), wild type currents at -120 mV were increased by 93.7% and 79.0%, respectively. Coapplication of the PKA inhibitor KT-5720 (2.5 micromol/l) attenuated this effect. No significant changes were apparent after mutation of the single PKA consensus site S430. In addition, removal of all four PKC consensus sites in Kir2.2 induced a phorbolester-mediated current increase which could be suppressed by PKA inhibitors H-89 (50 micromol/l) and KT-5720 (2.5 micromol/l)., Conclusions: This study demonstrates antagonistic effects of PKA and PKC in the regulation of Kir2.2. Phosphorylation by PKC has been shown to cause an inhibition of Kir2.2 currents, whereas activation of PKA leads to current upregulation., (Copryright 2004 European Society of Cardiology)

Objective: Adrenergic stimulation of the heart leads to activation of the phospholipase D signal transduction pathway with formation of the intracellular second messengers phosphatidic acid and diacylglycerol, which may play a role in the development of myocardial hypertrophy by activating mitogen-activated protein kinases and protein kinase C. So far, the adrenergic receptor subtypes mediating activation of cardiac phospholipase D are not known., Methods: We developed an assay for determination of phospholipase D activity in the isolated perfused rat heart. Utilizing the phospholipase D specific transphosphatidylation reaction the stable product phosphatidylethanol (PEtOH) is formed in rat hearts perfused in the presence of 1% ethanol. Myocardial PEtOH formation was used as a marker of phospholipase D activity and was determined by HPLC and evaporative light-scattering detection (PEtOH microg/mg myocardial protein)., Results: Basal PEtOH formation in unstimulated hearts was 0.06+/-0.01 microg/mg. Stimulation of the hearts with norepinephrine resulted in a concentration-dependent phospholipase D activation with a maximum formation of PEtOH (0.17+/-0.01 microg/mg) at 100 micromol/l norepinephrine. The norepinephrine-induced increase in PLD activity was completely blocked by the alpha(1)-adrenoceptor antagonist prazosin and was unaffected by the beta-adrenoceptor antagonist propranolol. Further characterisation of alpha(1)-adrenoceptor subtypes with selective alpha(1)-adrenoceptor antagonists demonstrated a complete inhibition of the norepinephrine-induced phospholipase D activation by WB 4101 (alpha(1A)-selective: 0.06+/-0.01 microg/mg) and by BMY 7378 (alpha(1D)-selective: 0.07+/-0.01 microg/mg). In contrast, the alpha(1B)-adrenoceptor antagonist chloroethylclonidine had no inhibitory effect on norepinephrine-stimulated phospholipase D activity (0.14+/-0.01 microg/mg)., Conclusion: Adrenergic activation of the cardiac phospholipase D signal transduction pathway is mediated by alpha(1)-adrenoceptors. Here, the alpha(1A)-adrenoceptor subtype, but not the alpha(1B)-adrenoceptor are coupled to activation of cardiac phospholipase D.