Diltiazem prevents stress-induced contractile deficits in cardiomyocytes, but does not reverse the cardiomyopathy phenotype in Mybpc3-knock-in mice.

KEY POINTS: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac illness and can lead to diastolic dysfunction, sudden cardiac death and heart failure. Treatment of HCM patients is empirical and current pharmacological treatments are unable to stop disease progression or reverse hypertrophy. In this study, we tested if the non-dihydropyridine Ca2+ channel blocker diltiazem, which previously showed potential to stop disease progression, can improve the phenotype of a HCM mouse model (Mybpc3-targeted knock-in), which is based on a mutation commonly found in patients. Diltiazem improved contractile function of isolated ventricular cardiomyocytes acutely, but chronic application did not improve the phenotype of adult mice with a fully developed HCM. Our study shows that diltiazem has beneficial effects in HCM, but long-term treatment success is likely to depend on characteristics and cause of HCM and onset of treatment. ABSTRACT: Left ventricular hypertrophy, diastolic dysfunction and fibrosis are the main features of hypertrophic cardiomyopathy (HCM). Guidelines recommend ß-adrenoceptor or Ca2+ channel antagonists as pharmacological treatment. The Ca2+ channel blocker diltiazem recently showed promising beneficial effects in pre-clinical HCM, particularly in patients carrying MYBPC3 mutations. In the present study we evaluated whether diltiazem could ameliorate or reverse the disease phenotype in cells and in vivo in an Mybpc3-targeted knock-in (KI) mouse model of HCM. Sarcomere shortening and Ca2+ transients were measured in KI and wild-type (WT) cardiomyocytes in basal conditions (1-Hz pacing) and under stress conditions (30 nm isoprenaline, 5-Hz pacing) with or without pre-treatment with 1 µm diltiazem. KI cardiomyocytes exhibited lower diastolic sarcomere length (dSL) at baseline, a tendency to a stronger positive inotropic response to isoprenaline than WT, a marked reduction of dSL and a tendency towards arrhythmias under stress conditions. Pre-treatment of cardiomyocytes with 1 µm diltiazem reduced the drop in dSL and arrhythmia frequency in KI, and attenuated the positive inotropic effect of isoprenaline. Furthermore, diltiazem reduced the contraction amplitude at 5 Hz but did not affect diastolic Ca2+ load and Ca2+ transient amplitude. Six months of diltiazem treatment of KI mice did not reverse cardiac hypertrophy and dysfunction, activation of the fetal gene program or fibrosis. In conclusion, diltiazem blunted the response to isoprenaline in WT and KI cardiomyocytes and improved diastolic relaxation under stress conditions in KI cardiomyocytes. This beneficial effect of diltiazem in cells did not translate in therapeutic efficacy when applied chronically in KI mice.

Comparison of the effects of a truncating and a missense MYBPC3 mutation on contractile parameters of engineered heart tissue.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. The most frequently mutated gene is MYBPC3, encoding cardiac myosin-binding protein-C (cMyBP-C). We compared the pathomechanisms of a truncating mutation (c.2373_2374insG) and a missense mutation (c.1591G>C) in MYBPC3 in engineered heart tissue (EHT). EHTs enable to study the direct effects of mutants without interference of secondary disease-related changes. EHTs were generated from Mybpc3-targeted knock-out (KO) and wild-type (WT) mouse cardiac cells. MYBPC3 WT and mutants were expressed in KO EHTs via adeno-associated virus. KO EHTs displayed higher maximal force and sensitivity to external [Ca(2+)] than WT EHTs. Expression of WT-Mybpc3 at MOI-100 resulted in ~73% cMyBP-C level but did not prevent the KO phenotype, whereas MOI-300 resulted in ≥95% cMyBP-C level and prevented the KO phenotype. Expression of the truncating or missense mutation (MOI-300) or their combination with WT (MOI-150 each), mimicking the homozygous or heterozygous disease state, respectively, failed to restore force to WT level. Immunofluorescence analysis revealed correct incorporation of WT and missense, but not of truncated cMyBP-C in the sarcomere. In conclusion, this study provides evidence in KO EHTs that i) haploinsufficiency affects EHT contractile function if WT cMyBP-C protein levels are ≤73%, ii) missense or truncating mutations, but not WT do not fully restore the disease phenotype and have different pathogenic mechanisms, e.g. sarcomere poisoning for the missense mutation, iii) the direct impact of (newly identified) MYBPC3 gene variants can be evaluated.

The E3 ubiquitin ligase Asb2ß is downregulated in a mouse model of hypertrophic cardiomyopathy and targets desmin for proteasomal degradation.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease with mutations in genes encoding sarcomeric proteins. Previous findings suggest deregulation of the ubiquitin proteasome system (UPS) in HCM in humans and in a mouse model of HCM (Mybpc3-targeted knock-in (KI) mice). In this study we investigated transcript levels of several muscle-specific E3 ubiquitin ligases in KI mice and aimed at identifying novel protein targets. METHODS AND RESULTS: Out of 9 muscle-specific E3 ligases, Asb2ß was found with the lowest mRNA level in KI compared to wild-type (WT) mice. After adenoviral-mediated Asb2ß transduction of WT neonatal mouse cardiomyocytes with either a WT or inactive Asb2ß mutant, desmin was identified as a new target of Asb2ß by mass spectrometry, co-immunoprecipitation and immunoblotting. Immunofluorescence analysis revealed a co-localization of desmin with Asb2ß at the Z-disk of the sarcomere. Knock-down of Asb2ß in cardiomyocytes resulted in higher desmin protein levels. Furthermore, desmin levels were higher in ventricular samples of HCM mice and patients than controls. CONCLUSIONS: This study identifies desmin as a new Asb2ß target for proteasomal degradation in cardiomyocytes and suggests that accumulation of desmin could contribute to UPS impairment in HCM mice and patients.

Evidence for FHL1 as a novel disease gene for isolated hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy, diastolic dysfunction and myocardial disarray. HCM is caused by mutations in sarcomeric genes, but in >40% of patients, the mutation is not yet identified. We hypothesized that FHL1, encoding four-and-a-half-LIM domains 1, could be another disease gene since it has been shown to cause distinct myopathies, sometimes associated with cardiomyopathy. We evaluated 121 HCM patients, devoid of a mutation in known disease genes. We identified three novel variants in FHL1 (c.134delA/K45Sfs, c.459C>A/C153X and c.827G>C/C276S). Whereas the c.459C>A variant was associated with muscle weakness in some patients, the c.134delA and c.827G>C variants were associated with isolated HCM. Gene transfer of the latter variants in C2C12 myoblasts and cardiac myocytes revealed reduced levels of FHL1 mutant proteins, which could be rescued by proteasome inhibition. Contractility measurements after adeno-associated virus transduction in rat-engineered heart tissue (EHT) showed: (i) higher and lower forces of contraction with K45Sfs and C276S, respectively, and (ii) prolonged contraction and relaxation with both mutants. All mutants except one activated the fetal hypertrophic gene program in EHT. In conclusion, this study provides evidence for FHL1 to be a novel gene for isolated HCM. These data, together with previous findings of proteasome impairment in HCM, suggest that FHL1 mutant proteins may act as poison peptides, leading to hypertrophy, diastolic dysfunction and/or altered contractility, all features of HCM.

Automated analysis of contractile force and Ca2+ transients in engineered heart tissue.

Contraction and relaxation are fundamental aspects of cardiomyocyte functional biology. They reflect the response of the contractile machinery to the systolic increase and diastolic decrease of the cytoplasmic Ca(2+) concentration. The analysis of contractile function and Ca(2+) transients is therefore important to discriminate between myofilament responsiveness and changes in Ca(2+) homeostasis. This article describes an automated technology to perform sequential analysis of contractile force and Ca(2+) transients in up to 11 strip-format, fibrin-based rat, mouse, and human fura-2-loaded engineered heart tissues (EHTs) under perfusion and electrical stimulation. Measurements in EHTs under increasing concentrations of extracellular Ca(2+) and responses to isoprenaline and carbachol demonstrate that EHTs recapitulate basic principles of heart tissue functional biology. Ca(2+) concentration-response curves in rat, mouse, and human EHTs indicated different maximal twitch forces (0.22, 0.05, and 0.08 mN in rat, mouse, and human, respectively; P < 0.001) and different sensitivity to external Ca(2+) (EC50: 0.15, 0.39, and 1.05 mM Ca(2+) in rat, mouse, and human, respectively; P < 0.001) in the three groups. In contrast, no difference in myofilament Ca(2+) sensitivity was detected between skinned rat and human EHTs, suggesting that the difference in sensitivity to external Ca(2+) concentration is due to changes in Ca(2+) handling proteins. Finally, this study confirms that fura-2 has Ca(2+) buffering effects and is thereby changing the force response to extracellular Ca(2+).

FHL2 expression and variants in hypertrophic cardiomyopathy.

Based on evidence that FHL2 (four and a half LIM domains protein 2) negatively regulates cardiac hypertrophy we tested whether FHL2 altered expression or variants could be associated with hypertrophic cardiomyopathy (HCM). HCM is a myocardial disease characterized by left ventricular hypertrophy, diastolic dysfunction and increased interstitial fibrosis and is mainly caused by mutations in genes coding for sarcomeric proteins. FHL2 mRNA level, FHL2 protein level and I-band-binding density were lower in HCM patients than control individuals. Screening of 121 HCM patients without mutations in established disease genes identified 2 novel (T171M, V187L) and 4 known (R177Q, N226N, D268D, P273P) FHL2 variants in unrelated HCM families. We assessed the structural and functional consequences of the nonsynonymous substitutions after adeno-associated viral-mediated gene transfer in cardiac myocytes and in 3D-engineered heart tissue (EHT). Overexpression of FHL2 wild type or nonsynonymous substitutions in cardiac myocytes markedly down-regulated α-skeletal actin and partially blunted hypertrophy induced by phenylephrine or endothelin-1. After gene transfer in EHTs, force and velocity of both contraction and relaxation were higher with T171M and V187L FHL2 variants than wild type under basal conditions. Finally, chronic phenylephrine stimulation depressed EHT function in all groups, but to a lower extent in T171M-transduced EHTs. These data suggest that (1) FHL2 is down-regulated in HCM, (2) both FHL2 wild type and variants partially protected phenylephrine- or endothelin-1-induced hypertrophy in cardiac myocytes, and (3) FHL2 T171M and V187L nonsynonymous variants induced altered EHT contractility. These findings provide evidence that the 2 novel FHL2 variants could increase cardiac function in HCM.

Localization of Islet-1-positive cells in the healthy and infarcted adult murine heart.

RATIONALE: The transcription factor Islet-1 is a marker of cardiovascular progenitors during embryogenesis. The isolation of Islet-1-positive (Islet-1(+)) cells from early postnatal hearts suggested that Islet-1 also marks cardiac progenitors in adult life. OBJECTIVE: We investigated the distribution and identity of Islet-1(+) cells in adult murine heart and evaluated whether their number or distribution change with age or after myocardial infarction. METHODS AND RESULTS: Distribution of Islet-1(+) cells in adult heart was investigated using gene targeted mice with nuclear ß-galactosidase inserted into the Islet-1 locus. nLacZ-positive cells were only present in 3 regions of the adult heart: clusters in the interatrial septum and around the pulmonary veins, scattered within the wall of the great vessels, and a strictly delimited cluster between the right atrium and superior vena cava. Islet-1(+) cells in the first type of clusters coexpressed markers for parasympathetic neurons. Positive cells in the great arteries coexpressed smooth muscle actin and myosin heavy chain, indicating a smooth muscle cell identity. Very few Islet-1(+) cells within the outflow tract expressed the cardiomyocyte marker α-actinin. Islet-1(+) cells in the right atrium coexpressed the sinoatrial node pacemaker cell marker HCN4. Cell number and localization remained unchanged between 1 to 18 months of age. Consistently Islet-1 mRNA was detected in human sinoatrial node. Islet-1(+) cells could not be detected in the infarct zone 2 to 28 days after myocardial infarction, aside from 10 questionable cells in 1/13 hearts. CONCLUSIONS: Our results identify Islet-1 as a novel marker of the adult sinoatrial node and do not provide evidence for Islet-1(+) cells to serve as cardiac progenitors.

Impact of COPD on mortality: An 8-year observational retrospective healthcare claims database cohort study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is one of the leading causes of morbidity and mortality. Here we present a large observational study on the association of COPD and exacerbations with mortality (AvoidEx Mortality). METHODS: A real-world, observational cohort study with longitudinal analyses of German healthcare claims data in patients ≥40 years of age with a COPD diagnosis from 2011 to 2018 (n = 250,723) was conducted. Patients entered the cohort (index date) upon the first COPD diagnosis. To assess the impact of COPD on all-cause death, a propensity score-matched control group of non-COPD patients was constructed. The number and severity of exacerbations during a 12-month pre-index period were used to form subgroups. For each exacerbation subgroup the exacerbations during 12 months prior to death were analysed. RESULTS: COPD increases the all-cause mortality risk by almost 60% (HR 1.57 (95% CI 1.55-1.59)) in comparison to matched non-COPD controls, when controlling for other baseline covariates. The cumulative risk of death after 8 years was highest in patients with a history of more than one moderate or severe exacerbation. Among all deceased COPD patients, 17.2% had experienced a severe, and 34.8% a moderate exacerbation, within 3 months preceding death. Despite increasing exacerbation rates towards death, more than the half of patients were not receiving any recommended pharmacological COPD therapy in the year before death. CONCLUSION: Our study illustrates the impact of COPD on mortality risk and highlights the need for consequent COPD management comprising exacerbation assessment and treatment.

Contractile abnormalities and altered drug response in engineered heart tissue from Mybpc3-targeted knock-in mice.

Myosin-binding protein C (Mybpc3)-targeted knock-in mice (KI) recapitulate typical aspects of human hypertrophic cardiomyopathy. We evaluated whether these functional alterations can be reproduced in engineered heart tissue (EHT) and yield novel mechanistic information on the function of cMyBP-C. EHTs were generated from cardiac cells of neonatal KI, heterozygous (HET) or wild-type controls (WT) and developed without apparent morphological differences. KI had 70% and HET 20% lower total cMyBP-C levels than WT, accompanied by elevated fetal gene expression. Under standard culture conditions and spontaneous beating, KI EHTs showed more frequent burst beating than WT and occasional tetanic contractions (14/96). Under electrical stimulation (6Hz, 37°C) KI EHTs exhibited shorter contraction and relaxation times and a twofold higher sensitivity to external [Ca(2+)]. Accordingly, the sensitivity to verapamil was 4-fold lower and the response to isoprenaline or the Ca(2+) sensitizer EMD 57033 2- to 4-fold smaller. The loss of EMD effect was verified in 6-week-old KI mice in vivo. HET EHTs were apparently normal under basal conditions, but showed similarly altered contractile responses to [Ca(2+)], verapamil, isoprenaline and EMD. In contrast, drug-induced changes in intracellular Ca(2+) transients (Fura-2) were essentially normal. In conclusion, the present findings in auxotonically contracting EHTs support the idea that cMyBP-C's normal role is to suppress force generation at low intracellular Ca(2+) and stabilize the power-stroke step of the cross bridge cycle. Pharmacological testing in EHT unmasked a disease phenotype in HET. The altered drug response may be clinically relevant.

COPD Exacerbation History and Impact on Future Exacerbations - 8-Year Retrospective Observational Database Cohort Study from Germany.

Background: Recent studies evaluating the predictive value of different variables on future exacerbations suggest exacerbation history as the strongest predictor. We examined the effect of exacerbation history on subsequent events in a large sample population with over 250,000 COPD patients using up to 8 years of longitudinal healthcare data from Germany. Methods: Patients 40 years or older with any COPD diagnosis in primary or secondary care were included from 2011 to 2017 (index period) from healthcare insurance claims (Germany; WIG2 research database), with 12 months before index date as baseline and at least 12-month follow-up. Exacerbations during baseline were defined as moderate (treatment with oral corticosteroids or antibiotics, J01AA, J01CA) or severe (emergency visit or hospitalization). Results: Patients without (category A), with one moderate (category B), or with either one severe or several baseline exacerbations (category C) experienced an average of 0.9 (CI 0.9-0.9), 1.9 (CI 1.9-1.9), and 6.3 (CI 6.1-6.3) exacerbations during the first 3 years of follow-up, respectively. By 8 years, 87.0% (CI 86.6-87.4), 70.5% (CI 69.9-71.0) and 49.1% (CI 48.9-49.3) of category C, B and A patients had experienced a subsequent exacerbation. Conclusion: Baseline exacerbations increased the likelihood of, and reduced time to subsequent exacerbations. Even patients without baseline exacerbations experienced exacerbations within three years, emphasizing the importance of adequate treatment in patients with less severe disease presentation as well.

Ouabain worsens diastolic sarcomere length in myocytes from a cardiomyopathy mouse model.

Diastolic dysfunction is a major feature of hypertrophic cardiomyopathy (HCM). Data from patient tissue and animal models associate increased Ca2+ sensitivity of myofilaments with altered Na+ and Ca2+ ion homeostasis in cardiomyocytes with diastolic dysfunction. In this study, we tested the acute effects of ouabain on ventricular myocytes of an HCM mouse model. The effects of ouabain on contractility and Ca2+ transients were tested in intact adult mouse ventricular myocytes (AMVMs) of Mybpc3-targeted knock-in (KI) and wild-type (WT) mice. Concentration-response assessment of contractile function revealed low sensitivity of AMVMs to ouabain (10 µM) compared to literature data on human cardiomyocytes (100 nM). Three hundred µM ouabain increased contraction amplitude (WT ~1.8-fold; KI ~1.5-fold) and diastolic intracellular Ca2+ in both WT and KI (+12-18%), but further decreased diastolic sarcomere length in KI cardiomyocytes (-5%). Western Blot analysis of whole heart protein extracts revealed 50% lower amounts of Na+/K+ ATPase (NKA) in KI than in WT. Ouabain worsened the diastolic phenotype of KI cardiomyocytes at concentrations which did not impair WT diastolic function. Ouabain led to an elevation of intracellular Ca2+, which was poorly tolerated in KI showing already high cytosolic Ca2+ at baseline due to increased myofilament Ca2+ sensitivity. Lower amounts of NKA in KI could amplify the need to exchange excessive intracellular Na+ for Ca2+ and thereby explain the general tendency to higher diastolic Ca2+ in KI.

Sulforaphane exposure impairs contractility and mitochondrial function in three-dimensional engineered heart tissue.

Sulforaphane (SFN) is a phytochemical compound extracted from cruciferous plants, like broccoli or cauliflower. Its isothiocyanate group renders SFN reactive, thus allowing post-translational modification of cellular proteins to regulate their function with the potential for biological and therapeutic actions. SFN and stabilized variants recently received regulatory approval for clinical studies in humans for the treatment of neurological disorders and cancer. Potential unwanted side effects of SFN on heart function have not been investigated yet. The present study characterizes the impact of SFN on cardiomyocyte contractile function in cardiac preparations from neonatal rat, adult mouse and human induced-pluripotent stem cell-derived cardiomyocytes. This revealed a SFN-mediated negative inotropic effect, when administered either acutely or chronically, with an impairment of the Frank-Starling response to stretch activation. A direct effect of SFN on myofilament function was excluded in chemically permeabilized mouse trabeculae. However, SFN pretreatment increased lactate formation and enhanced the mitochondrial production of reactive oxygen species accompanied by a significant reduction in the mitochondrial membrane potential. Transmission electron microscopy revealed disturbed sarcomeric organization and inflated mitochondria with whorled membrane shape in response to SFN exposure. Interestingly, administration of the alternative energy source l-glutamine to the medium that bypasses the uptake route of pyruvate into the mitochondrial tricarboxylic acid cycle improved force development in SFN-treated EHTs, suggesting indeed mitochondrial dysfunction as a contributor of SFN-mediated contractile dysfunction. Taken together, the data from the present study suggest that SFN might impact negatively on cardiac contractility in patients with cardiovascular co-morbidities undergoing SFN supplementation therapy. Therefore, cardiac function should be monitored regularly to avoid the onset of cardiotoxic side effects.

A new polymorphism in human calmodulin III gene promoter is a potential modifier gene for familial hypertrophic cardiomyopathy.

AIMS: Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in genes encoding sarcomeric proteins. Incomplete penetrance suggests the existence of modifier genes. Calmodulin (CaM) could be of importance given the key role of Ca(2+) for cardiac contractile function and growth. Any variant that affects CaM expression and/or function may impact on FHC clinical expression. METHODS AND RESULTS: We screened the promoter region of human calmodulin III gene (CALM3) and identified a new -34T>A polymorphism with a T-allele frequency of 0.70. The distribution of CALM3 genotypes differed in 180 unrelated FHC patients carrying a known FHC mutation compared with 134 controls, with higher TT-genotype frequency (0.73 vs. 0.51) and lower frequencies of AT- (0.24 vs. 0.37) and AA genotypes (0.03 vs. 0.11; P = 0.0005). To study whether the -34T>A polymorphism could play a modifier role, patients' relatives including both affected and healthy carriers were added. Affected carriers had a 0.56 times higher odds of carrying a T allele than healthy carriers (P = 0.053). We then investigated whether the -34T>A polymorphism affects the promoter activity using luciferase reporter vectors containing either CALM3-T or CALM3-A promoters. The activity of CALM3-T was lower than CALM3-A in HEK293 cells (1.00 +/- 0.19 vs. 2.31 +/- 0.13, P = 0.00001) and in cardiomyocytes (0.96 +/- 0.10 vs. 1.33 +/- 0.08, P = 0.00727). CONCLUSION: These data suggest that the -34T>A CALM3 polymorphism is a modifier gene for FHC, potentially by affecting expression level of CALM3 and therefore Ca(2+)-handling and development of hypertrophy.

Effectiveness and Tolerability of LABA/LAMA Fixed-Dose Combinations Aclidinium/Formoterol, Glycopyrronium/Indacaterol and Umeclidinium/Vilanterol in the Treatment of COPD in Daily Practice - Results of the Non-Interventional DETECT Study.

Background: LABA (long-acting ß2-agonists) and/or LAMA (long-acting muscarinic antagonists) represent the first treatment options for patients with symptomatic COPD. Although both display different mechanisms of activity, in combination they have a stronger broncho-dilating effect than monotherapy; hence, a combination of both LABA and LAMA is particularly recommended for patients whose symptoms cannot be sufficiently improved by a single active ingredient. To date, only few data have been collected regarding the therapeutic outcomes of approved LABA/LAMA fixed-dose combinations (FDCs) under everyday (real-life) conditions in non-clinical trial settings. Objective and Methods: The main objective of the DETECT study was to investigate the impact of aclidinium/formoterol (AB/FF, b.i.d.), glycopyrronium/indacaterol (GLY/IND, q.d.) and umeclidinium/vilanterol (UME/VL, q.d.) in patients with COPD in daily clinical practice. Therefore, a prospective, non-randomized, 12-month, observational study was implemented to assess the effectiveness of these treatments in patients who had been switched to FDC within the last 3 months or for whom such a changeover was intended. Changes in lung function were analyzed by the forced expiratory volume (FEV1) and forced vital capacity (FVC) measures. Quality of life and well-being were evaluated by the COPD Assessment Test (CAT™). Furthermore, a number of exacerbations and early morning COPD symptoms were documented. Results: In total, 3653 patients were enrolled. FEV1 and FVC values significantly improved during the study with AB/FF (increase by 0.09 ± 0.40 L and 0.10 ± 0.57 L, respectively; p<0.0001), GLY/IND (0.06±0.38/0.05±0.51 L; p<0.0001 and p=0.0025) and UME/VL (0.12±0.39/0.10±0.52 L; p<0.0001). CAT scores decreased indicating improved COPD (AB/FF, 4.17±8.30; GLY/IND, 3.66±7.88; UME/VL, 4.06±7.96; p<0.0001). Moreover, the number of exacerbations as well as early morning COPD symptoms similarly diminished in all treatment groups. A comparable proportion of patients with adverse drug reactions was recorded: AB/FF, 4.07% of patients; GLY/IND, 3.52%; UME/VL, 3.64%. Conclusion: In summary, AB/FF, GLY/IND and UME/VL provided clinical benefits in lung function, quality of life and early morning COPD symptoms in a broad cohort of COPD patients under routine medical practice conditions. All three treatments were well tolerated.

Effect of ranolazine on plasma arginine derivatives and urinary isoprostane 8-iso-PGF2α in patients with myocardial infarction in the randomized RIMINI-Trial.

The purpose of the present study was to assess whether 6-week ranolazine application on top of guideline-based treatment impacts on the arginine/NO pathway and urinary isoprostane 8-iso-PGF2α as marker of oxidative stress in patients directly after a myocardial infarction. 20 patients with unstable angina pectoris and proof of acute cardiac ischemia entered the study. 10 subjects received the study drug ranolazine in addition to standard treatment, the others received only standard treatment. Urine and venous blood were collected before and after treatment. At the end of the study and compared to baseline, homoarginine levels had increased in the control group. This was not the case in ranolazine-patients. Interestingly, in ranolazine-treated-patients arginine plasma levels were significantly higher at the end of the study than at baseline (difference +26 µmol/L, 95% CI 8.6 to 44 µmol/L). ADMA and SDMA levels were not different. Urine levels of the oxidative stress marker 8-iso-PGF2α tended to be lower in ranolazine-treated patients (-144 pmol/mg creatinine). Findings of this hypothesis-driven study give evidence that ranolazine treatment enhances arginine plasma levels and lowers oxidative stress.

Effect of Ranolazine on Ischemic Myocardium IN Patients With Acute Cardiac Ischemia (RIMINI-Trial): A Randomized Controlled Pilot Trial.

BACKGROUND: Coronary artery disease is the most prevalent manifestation among cardiovascular diseases. Despite modern treatment, risk of ischemic complications in patients with acute coronary syndrome (ACS) remains important. The late Na+ current blocker ranolazine has shown to reduce the risk of recurrent ischemia and worsening of angina in patients with non-ST-segment elevation ACS by possibly improving myocardial perfusion, but up to now no trial has addressed whether this enhanced perfusion also leads to a decrease in ischemic myocardium of patients with ACS. We designed a pilot trial (Reduction of Ischemic Myocardium with Ranolazine-Treatment IN patients with acute myocardial Infarction, ClinicalTrials.gov Identifier: NCT01797484) for feasibility and proof of concept that a 6-week ranolazine add-on therapy would reduce the area of ischemic myocardium in patients with ACS. METHODS AND RESULTS: The trial was designed in a 2-armed, controlled and randomized way. Twenty participants with unstable angina, proof of acute cardiac ischemia, and myocardial dyskinesia by speckle-tracking echocardiography were included. Ten participants received the study drug ranolazine additionally to standard treatment. The control group received standard treatment without additional study medication. Speckle-tracking echocardiography was performed before coronary intervention, before the first dose of ranolazine, and after 6 weeks of ranolazine treatment. Ranolazine was administered safely during acute myocardial infarction. Speckle-tracking echocardiography proved to be suitable for evaluation of myocardial dyskinesia. Patients receiving ranolazine showed a trend to higher normal fraction of the cumulative global strain than patients in the standard treatment group (15% vs 11%). No major complications relating study medication were observed. CONCLUSION: In conclusion, in this preliminary hypothesis-driven study, 6-week ranolazine therapy was shown to decrease the area of dyskinetic myocardium in patients with ACS by trend. Global strain rate measurement using speckle-tracking echocardiography can be applied measuring those effects and is, compared to other techniques, safe and harmless. Our data provide a sound basis for a follow-up trial.

Phosphomimetic cardiac myosin-binding protein C partially rescues a cardiomyopathy phenotype in murine engineered heart tissue.

Phosphorylation of cardiac myosin-binding protein C (cMyBP-C), encoded by MYBPC3, increases the availability of myosin heads for interaction with actin thus enhancing contraction. cMyBP-C phosphorylation level is lower in septal myectomies of patients with hypertrophic cardiomyopathy (HCM) than in non-failing hearts. Here we compared the effect of phosphomimetic (D282) and wild-type (S282) cMyBP-C gene transfer on the HCM phenotype of engineered heart tissues (EHTs) generated from a mouse model carrying a Mybpc3 mutation (KI). KI EHTs showed lower levels of mutant Mybpc3 mRNA and protein, and altered gene expression compared with wild-type (WT) EHTs. Furthermore, KI EHTs exhibited faster spontaneous contractions and higher maximal force and sensitivity to external [Ca2+] under pacing. Adeno-associated virus-mediated gene transfer of D282 and S282 similarly restored Mybpc3 mRNA and protein levels and suppressed mutant Mybpc3 transcripts. Moreover, both exogenous cMyBP-C proteins were properly incorporated in the sarcomere. KI EHTs hypercontractility was similarly prevented by both treatments, but S282 had a stronger effect than D282 to normalize the force-Ca2+-relationship and the expression of dysregulated genes. These findings in an in vitro model indicate that S282 is a better choice than D282 to restore the HCM EHT phenotype. To which extent the results apply to human HCM remains to be seen.

Disease modeling of a mutation in α-actinin 2 guides clinical therapy in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease accompanied by structural and contractile alterations. We identified a rare c.740C>T (p.T247M) mutation in ACTN2, encoding α-actinin 2 in a HCM patient, who presented with left ventricular hypertrophy, outflow tract obstruction, and atrial fibrillation. We generated patient-derived human-induced pluripotent stem cells (hiPSCs) and show that hiPSC-derived cardiomyocytes and engineered heart tissues recapitulated several hallmarks of HCM, such as hypertrophy, myofibrillar disarray, hypercontractility, impaired relaxation, and higher myofilament Ca2+ sensitivity, and also prolonged action potential duration and enhanced L-type Ca2+ current. The L-type Ca2+ channel blocker diltiazem reduced force amplitude, relaxation, and action potential duration to a greater extent in HCM than in isogenic control. We translated our findings to patient care and showed that diltiazem application ameliorated the prolonged QTc interval in HCM-affected son and sister of the index patient. These data provide evidence for this ACTN2 mutation to be disease-causing in cardiomyocytes, guiding clinical therapy in this HCM family. This study may serve as a proof-of-principle for the use of hiPSC for personalized treatment of cardiomyopathies.

S100A4 as a Target of the E3-Ligase Asb2ß and Its Effect on Engineered Heart Tissue.

Background: S100A4 has recently emerged as an important player in cardiac disease, affecting phenotype development in animal models of myocardial infarction and pathological cardiac hypertrophy, albeit it is unclear whether S100A4 exerts a detrimental or beneficial function. The goal of the current study was to analyze S100A4 expression in models of cardiac pathology, investigate its degradation by the ubiquitin-proteasome system (UPS), and furthermore examine the functional effects of S100A4 levels in a 3D model of engineered heart tissue (EHT). Methods and Results: S100A4 mRNA and protein levels were analyzed in different models of cardiac pathology via quantitative RT-PCR and Western blot, showing a higher S100A4 steady-state protein concentration in hearts of Mybpc3-knock-in (KI) hypertrophic cardiomyopathy (HCM) mice. COS-7 cells co-transfected with plasmids encoding mutant (MUT) Asb2ß lacking the E3 ligase activity in combination with V5-tagged S100A4 plasmid presented higher S100A4-V5 protein steady-state concentrations than cells co-transfected with the Asb2ß wild type (WT) plasmid. This effect was blunted by treatment with the specific proteasome inhibitor epoxomicin. Adeno-associated virus serotype 6 (AAV6)-mediated S100A4 overexpression in a 3D model of EHT did not affect contractile parameters. Immunofluorescence analysis showed a cytosolic and partly nuclear expression pattern of S100A4. Gene expression analysis in EHTs overexpressing S100A4-V5 showed markedly lower steady-state concentrations of genes involved in cardiac fibrosis and pathological cardiac hypertrophy. Conclusion: We showed that S100A4 protein level is higher in cardiac tissue of Mybpc3-KI HCM mice probably as a result of a lower degradation by the E3 ligase Asb2ß. While an overexpression of S100A4 did not alter contractile parameters in EHTs, downstream gene expression analysis points toward modulation of signaling cascades involved in fibrosis and hypertrophy.

Nebivolol Desensitizes Myofilaments of a Hypertrophic Cardiomyopathy Mouse Model.

Background: Hypertrophic cardiomyopathy (HCM) patients often present with diastolic dysfunction and a normal to supranormal systolic function. To counteract this hypercontractility, guideline therapies advocate treatment with beta-adrenoceptor and Ca2+ channel blockers. One well established pathomechanism for the hypercontractile phenotype frequently observed in HCM patients and several HCM mouse models is an increased myofilament Ca2+ sensitivity. Nebivolol, a commonly used beta-adrenoceptor antagonist, has been reported to lower maximal force development and myofilament Ca2+ sensitivity in rabbit and human heart tissues. The aim of this study was to evaluate the effect of nebivolol in cardiac muscle strips of an established HCM Mybpc3 mouse model. Furthermore, we investigated actions of nebivolol and epigallocatechin-gallate, which has been shown to desensitize myofilaments for Ca2+ in mouse and human HCM models, in cardiac strips of HCM patients with a mutation in the most frequently mutated HCM gene MYBPC3. Methods and Results: Nebivolol effects were tested on contractile parameters and force-Ca2+ relationship of skinned ventricular muscle strips isolated from Mybpc3-targeted knock-in (KI), wild-type (WT) mice and cardiac strips of three HCM patients with MYBPC3 mutations. At baseline, KI strips showed no difference in maximal force development compared to WT mouse heart strips. Neither 1 nor 10 µM nebivolol had an effect on maximal force development in both genotypes. 10 µM nebivolol induced myofilament Ca2+ desensitization in WT strips and to a greater extent in KI strips. Neither 1 nor 10 µM nebivolol had an effect on Ca2+ sensitivity in cardiac muscle strips of three HCM patients with MYBPC3 mutations, whereas epigallocatechin-gallate induced a right shift in the force-Ca2+ curve. Conclusion: Nebivolol induced a myofilament Ca2+ desensitization in both WT and KI strips, which was more pronounced in KI muscle strips. In human cardiac muscle strips of three HCM patients nebivolol had no effect on myofilament Ca2+ sensitivity.