Cardiomyocyte Contractile Properties Research Articles

Pathophysiological role of epicardial adipose tissue and its phenotypes in patients with diabetes and cardiovascular comorbidities

Abstract Introduction Epicardial adipose tissue (EAT) is an active endocrine organ that regulates multiple aspects of cardiac biology including myocardial redox state, intracellular Ca2+ cycling, the electrophysiological and contractile properties of cardiomyocytes, cardiac fibrosis, and coronary atherosclerosis progression. Purpose We evaluated associations of CMR-measured EAT area (EATa) with myocardial structure, function and 31P-MRS assessed energetics (phosphocreatine to ATP ratio) in patients with type 2 diabetes (T2D) with and without comorbidities of coronary artery disease (CAD) requiring coronary artery bypass grafting (CABG) and aortic stenosis requiring aortic valve replacement (AVR). We also examined the EAT phenotypes in patients with T2D with and without CAD, CAD patients without T2D and compared them to controls with severe AS without T2D. Methods 145 participants were recruited including CAD patients undergoing CABG with (30) and without T2D (25); severe AS patients with (20) and without T2D (20) undergoing AVR; T2D patients without cardiovascular disease (30) and healthy controls (20). A month prior to surgery cardiac PCr/ATP; EATa; LV volumes, mass, ejection fraction, mitral inflow E/A ratio, global longitudinal strain (GLS) and myocardial tissue characteristics were assessed. EAT samples were obtained from patients undergoing cardiac surgery for histological assessments. Results Participants were matched in age, sex distribution, and body mass index. Across all groups, patients with T2D showed higher EATa compared to those without T2D (138±50 vs 101±51cm2; p<0.0001). EATa was significantly higher in AS patients with T2D versus AS patients without T2D, while there was no significant difference in EATa between the CAD patients with and without T2D. Among the structural parameters, there were significant correlations between the EATa and myocardial pre-contrast T1 (p=0.02; r=0.20). Among the functional parameters there were inverse correlations of EATa with mitral inflow E/A (r=-0.34, p=0.002) and with GLS (r=-0.23, p=0.009). Among the metabolic biomarkers, there were no correlations between the EATa and PCr/ATP ratio. Histologically, compared to the control group of AS patients without T2D, patients with CAD with and without T2D showed larger adipocyte size and higher tissue fibrosis. There were no significant differences in EAT phenotype in adipocyte size, tissue fibrosis or inflammation between CAD patients with and without T2D. Conclusions This comprehensive EAT phenotyping study combined invasive and non-invasive methodologies and showed similar magnitudes of changes in EATa, adipocyte size and EAT fibrosis in CAD patients with and without T2D. While we have detected significant relationship of the EATa with the myocardial fibrosis, diastolic function and GLS, there were no correlations of myocardial PCr/ATP with the EATa suggesting other key mechanisms involved in the energetic deficit in patients with T2D.

Abstract 13073: Antisense Transcripts Regulate Titin Alternative Splicing and Sarcomere Function in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

The giant protein Titin is an essential structural component of the sarcomere and a key determinant of passive tension during diastole. Titin tension is regulated by alternative splicing within the spring-like domains in the I-band region of TTN pre-mRNA. The cardiac RNA-binding protein RBM20 has been shown to promote TTN exon skipping during splicing, but additional factors are likely involved. We hypothesized that a natural antisense transcript in the TTN locus ( TTN-AS1 ) could regulate TTN splicing in cis through recruitment of spliceosome components. Through single nuclei RNA-Seq and RNA fluorescence in situ hybridization (ISH) on human cardiac tissue and iPS-derived cardiomyocytes (iPS-CM), we showed that the expression of TTN-AS1 was restricted to cardiomyocyte nuclei. The percent spliced in (PSI) of each TTN exon was calculated based on RNA-Seq data from iPS-CM (n=3) and 26 exons (all in the I-band region) that are normally spliced out were included to a significantly higher extent (adjusted p<0.05) upon siRNA-mediated TTN-AS1 knock down. Reduced exon skipping from TTN exon 50 was confirmed with exon-junction spanning qPCR probes (p<0.05, n=3). Similar effects on splicing were seen in cells where RBM20 had been knocked down. The consequences of TTN-AS1 knock down on cardiomyocyte contractile function was assessed using sarcomere tracking. Transgenic expression of ACTN2-GFP was used to visualize z-disks in iPS-CM, sarcomeres (n=14,636) were recorded during contractions using wide-field epifluorescence microscopy and analyzed using Sarc-Graph software. The analysis revealed that in cells where TTN-AS1 had been knocked down, sarcomere length and fractional shortening, as well as contraction and relaxation time were increased (all p<0.001). Again, similar effects were seen in iPS-CM where RBM20 had been knocked down. We observed co-localization of TTN-AS1 and RBM20 protein during active TTN splicing in iPS-CM nuclei using multiplex RNA ISH and immunofluorescence and confirmed interaction between TTN-AS1 and RBM20 using RNA Immunoprecipitation and qPCR. In conclusion, we show that TTN-AS1 can affect cardiomyocyte contractile properties and sarcomere dynamics through regulation of TTN alternative splicing.

Abstract 11004: Activated Protein C Protects Human Cardiomyocytes Against Hypoxia Reoxygenation Stress-Induced Injury Through Maintaining Mitochondrial Integrity

Introduction: Activated protein C (APC) is a vitamin-K-dependent plasma serine protease that functions as a natural anticoagulant to downregulate thrombin generation in the clotting cascade. Endothelial protein C receptor (EPCR) mediates APC's cytoprotective properties. We found that cardiac EPCR expression is downregulated in aging, leading to cardiac intolerance to ischemic insults. However, the role of APC in the human heart under ischemic stress remains unclear. Hypothesis: APC-EPCR axis protects human cardiomyocytes against hypoxia-reoxygenation stress-induced damage through maintaining mitochondrial integrity. Methods: The left ventricles of 38 adult human donor hearts underwent proteomic analysis of myocardial EPCR. Viable human cardiomyocytes were isolated and challenged by 30 min hypoxia/30 min reoxygenation (H/R) with or without APC treatment. Cardiomyocyte contractile properties were determined by IonOptix Imaging System, while the mitochondrial functional integrity and reactive oxygen species (ROS) were determined with Seahorse XF analyzer and MitoSox staining. Results: Immunoblotting data demonstrated that EPCR expression levels are declined in human hearts along with aging. H/R-stress reduced contractile functions of human cardiomyocytes, and the cardiomyocytes from aged adults (>65 years) versus cardiomyocytes from young adults (<65 years) were more vulnerable to H/R-induced insults. Interestingly, the administration of APC significantly improved the contractile functions of both young and aged human cardiomyocytes under H/R stress. APC also stabilizes EPCR levels and modulates the calcium flux homeostasis in both young and aged human cardiomyocytes under H/R. The mitochondrial respiration experimental data demonstrated that APC treatment ameliorates the mitochondrial dysfunction induced by H/R stress in both young and aged human cardiomyocytes. Since the ROS generations in human cardiomyocytes during H/R stress were attenuated with APC administration. Conclusions: Cardiac EPCR levels are downregulated with aging. The administration of APC can rescue the cardiac intolerance to ischemic insults in aging through stabilizing receptor EPCR and maintaining cardiomyocyte mitochondrial integrity.

The ryanodine receptor stabilizer S107 ameliorates contractility of adult Rbm20 knockout rat cardiomyocytes.

RNA binding motif 20 (RBM20) cardiomyopathy has been detected in approximately 3% of populations afflicted with dilated cardiomyopathy (DCM). It is well conceived that RBM20 cardiomyopathy is provoked by titin isoform switching in combination with resting Ca2+ leaking. In this study, we characterized the cardiac function in Rbm20 knockout (KO) rats at 3‐, 6‐, 9‐, and 12‐months of age and examined the effect of the ryanodine receptor stabilizer S107 on resting intracellular levels and cardiomyocyte contractile properties. Our results revealed that even though Rbm20 depletion promoted expression of larger titin isoform and reduced myocardial stiffness in young rats (3 months of age), the established DCM phenotype required more time to embellish. S107 restored elevated intracellular Ca2+ to normal levels and ameliorated cardiomyocyte contractile properties in isolated cardiomyocytes from 6‐month‐old Rbm20 KO rats. However, S107 failed to preserve cardiac homeostasis in Rbm20 KO rats at 12 months of age, unexpectedly, likely due to the existence of multiple pathogenic mechanisms. Taken together, our data suggest the therapeutic promises of S107 in the management of RBM20 cardiomyopathy.

Sex-specific cardiac remodeling in early and advanced stages of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most frequent genetic cardiac disease with a prevalence of 1:500 to 1:200. While most patients show obstructive HCM and a relatively stable clinical phenotype (stage II), a small group of patients progresses to end-stage HCM (stage IV) within a relatively brief period. Previous research has shown sex-differences in stage II HCM with more diastolic dysfunction in female than in male patients. Moreover, female patients more often show progression to heart failure. Here we investigated if differences in functional and structural properties of the heart may underlie sex-differences in disease progression from stage II to stage IV HCM. Cardiac tissue from stage II and IV patients was obtained during myectomy (n = 54) and heart transplantation (n = 10), respectively. Isometric force was measured in membrane-permeabilized cardiomyocytes to define active and passive myofilament force development. Titin isoform composition was assessed using gel electrophoresis, and the amount of fibrosis and capillary density were determined with histology. In accordance with disease stage-dependent adverse cardiac remodeling end-stage patients showed a thinner interventricular septal wall and larger left ventricular and atrial diameters compared to stage II patients. Cardiomyocyte contractile properties and fibrosis were comparable between stage II and IV, while capillary density was significantly lower in stage IV compared to stage II. Women showed more adverse cellular remodeling compared to men at stage II, evident from more compliant titin, more fibrosis and lower capillary density. However, the disease stage-dependent reduction in capillary density was largest in men. In conclusion, the more severe cellular remodeling in female compared to male stage II patients suggests a more advanced disease stage at the time of myectomy in women. Changes in cardiomyocyte contractile properties do not explain the progression of stage II to stage IV, while reduced capillary density may underlie disease progression to end-stage heart failure.

Increased myocyte calcium sensitivity in end-stage pediatric dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is the most common cause of heart failure (HF) in children, resulting in high mortality and need for heart transplantation. The pathophysiology underlying pediatric DCM is largely unclear; however, there is emerging evidence that molecular adaptations and response to conventional HF medications differ between children and adults. To gain insight into alterations leading to systolic dysfunction in pediatric DCM, we measured cardiomyocyte contractile properties and sarcomeric protein phosphorylation in explanted pediatric DCM myocardium (N = 8 subjects) compared with nonfailing (NF) pediatric hearts (N = 8 subjects). Force-pCa curves were generated from skinned cardiomyocytes in the presence and absence of protein kinase A. Sarcomeric protein phosphorylation was quantified with Pro-Q Diamond staining after gel electrophoresis. Pediatric DCM cardiomyocytes demonstrate increased calcium sensitivity (pCa50 =5.70 ± 0.0291), with an associated decrease in troponin (Tn)I phosphorylation compared with NF pediatric cardiomyocytes (pCa50 =5.59 ± 0.0271, P = 0.0073). Myosin binding protein C and TnT phosphorylation are also lower in pediatric DCM, whereas desmin phosphorylation is increased. Pediatric DCM cardiomyocytes generate peak tension comparable to that of NF pediatric cardiomyocytes [DCM 29.7 mN/mm2, interquartile range (IQR) 21.5-49.2 vs. NF 32.8 mN/mm2, IQR 21.5-49.2 mN/mm2; P = 0.6125]. In addition, cooperativity is decreased in pediatric DCM compared with pediatric NF (Hill coefficient: DCM 1.56, IQR 1.31-1.94 vs. NF 1.94, IQR 1.36-2.86; P = 0.0425). Alterations in sarcomeric phosphorylation and cardiomyocyte contractile properties may represent an impaired compensatory response, contributing to the detrimental DCM phenotype in children.NEW & NOTEWORTHY Our study is the first to demonstrate that cardiomyocytes from infants and young children with dilated cardiomyopathy (DCM) exhibit increased calcium sensitivity (likely mediated by decreased troponin I phosphorylation) compared with nonfailing pediatric cardiomyocytes. Compared with published values in adult cardiomyocytes, pediatric cardiomyocytes have notably decreased cooperativity, with a further reduction in the setting of DCM. Distinct adaptations in cardiomyocyte contractile properties may contribute to a differential response to pharmacological therapies in the pediatric DCM population.

P27 Protects Cardiomyocytes from Sepsis via Activation of Autophagy and Inhibition of Apoptosis.

BackgroundIt has been reported that p27Kip1 plays an important role not only in the inhibition of cyclin-dependent kinases but also in the regulation of autophagy under various metabolically related stress conditions, including glucose deprivation and endoplasmic reticulum stress. However, its effect on lipopolysaccharide (LPS)-induced cardiomyocyte stress in vitro remains unclear. Here, we measured the increased expression of LC3-II and visualized autophagosomes in vitro by immunofluorescent assays after treatment with a p27 fusion protein.Material/MethodsCardiomyocyte contractile properties were assessed by measuring cell shortening and re-lengthening. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Western blot, colorectal ligation puncture (CLP) surgery, silencing of Atg5 expression by small interfering RNA (siRNA), and immunofluorescent assays were also performed in this study.ResultsAfter exogenous delivery of the p27 fusion protein and overexpression of p27 in LPS-induced cardiomyocytes, we found lower expressions of caspase-3 and caspase-8 and reduced positive TUNEL staining. Improved cardiomyocyte mechanical functions and reduced apoptosis were diminished after treatment with various autophagy inhibitors. Intravenous injections of p27-expressing adeno-associated virus serotype 9 (AAV9) vectors resulted in cardiac specific overexpression of p27, and echocardiography was used to assess cardiac function and structure in sepsis rat models. We observed improved cardiac function and reversed adverse ventricular remolding after the introduction of AAV9 vectors. Meanwhile, apoptosis was reduced, and expression of LC3-II was elevated in septic rat models treated with AAV9 vectors compared to controls.ConclusionsThe study data demonstrated that the overexpression of p27 protects cardiomyocytes from sepsis-induced cardiac depression via the activation of autophagy and inhibition of apoptosis.

Contractile properties of cardiomyocytes do not differ between obstructive and end-stage hypertrophic cardiomyopathy

Contractile properties of cardiomyocytes do not differ between obstructive and end-stage hypertrophic cardiomyopathy

In vivo administration of urolithin A and B prevents the occurrence of cardiac dysfunction in streptozotocin-induced diabetic rats

BackgroundEmerging evidence suggests that specific (poly)phenols may constitute new preventative strategies to counteract cell oxidative stress and myocardial tissue inflammation, which have a key role in the patho-physiology of diabetic cardiomyopathy. In a rat model of early diabetes, we evaluated whether in vivo administration of urolithin A (UA) or urolithin B (UB), the main gut microbiota phenolic metabolites of ellagitannin-rich foods, can reduce diabetes-induced microenvironmental changes in myocardial tissue, preventing cardiac functional impairment.MethodsAdult Wistar rats with streptozotocin-induced type-1 diabetes (n = 29) were studied in comparison with 10 control animals. Diabetic rats were either untreated (n = 9) or subjected to daily i.p. injection of UA (n = 10) or UB (n = 10). After 3 weeks of hyperglycaemia, hemodynamics, cardiomyocyte contractile properties and calcium transients were measured to assess cardiac performance. The myocardial expression of the pro-inflammatory cytokine fractalkine and proteins involved in calcium dynamics (sarcoplasmic reticulum calcium ATPase, phospholamban and phosphorylated phospholamban) were evaluated by immunoblotting. Plasma, urine and tissue distribution of UA, UB and their phase II metabolites were determined.ResultsIn vivo urolithin treatment reduced by approximately 30% the myocardial expression of the pro-inflammatory cytokine fractalkine, preventing the early inflammatory response of cardiac cells to hyperglycaemia. The improvement in myocardial microenvironment had a functional counterpart, as documented by the increase in the maximal rate of ventricular pressure rise compared to diabetic group (+18% and +31% in UA and UB treated rats, respectively), and the parallel reduction in the isovolumic contraction time (−12%). In line with hemodynamic data, both urolithins induced a recovery of cardiomyocyte contractility and calcium dynamics, leading to a higher re-lengthening rate (+21%, on average), lower re-lengthening times (−56%), and a more efficient cytosolic calcium clearing (−32% in tau values). UB treatment also increased the velocity of shortening (+27%). Urolithin metabolites accumulated in the myocardium, with a higher concentration of UB and UB-sulphate, potentially explaining the slightly higher efficacy of UB administration.ConclusionsIn vivo urolithin administration may be able to prevent the initial inflammatory response of myocardial tissue to hyperglycaemia and the negative impact of the altered diabetic milieu on cardiac performance.

Alcoholic Cardiomyopathy: Disrupted Protein Balance and Impaired Cardiomyocyte Contractility.

Alcoholic cardiomyopathy (ACM) can develop after consumption of relatively large amounts of alcohol over time or from acute binge drinking. Of the many factors implicated in the etiology of ACM, chronic perturbation in protein balance has been strongly implicated. This review focused on recent contributions (since 2010) in the area of protein metabolism and cardiac function related to ACM. Data reviewed include that from invitro and preclinical invivo animal studies where alcohol or an oxidative metabolite was studied and outcome measures in either cardiomyocytes or whole heart pertaining to protein synthesis or degradation were reported. Additionally, studies on the contractile properties of cardiomyocytes were also included to link signal transduction with function. Methodological differences including the potential impact of sex, dosing, and duration/timing of alcohol administration are addressed. Acute and chronic alcohol consumption decreases cardiac protein synthesis and/or activation of proteins within the regulatory mammalian/mechanistic target of rapamycin complex pathway. Albeit limited, evidence suggests that myocardial protein degradation via the ubiquitin pathway is not altered, while autophagy may be enhanced in ACM. Alcohol impairs exvivo cardiomyocyte contractility in relation to its metabolism and expression of proteins within the growth factor pathway. Dysregulation of protein metabolism, including the rate of protein synthesis and autophagy, may contribute to contractile deficits and is a hallmark feature of ACM meriting additional sex-inclusive, methodologically consistent studies.

The role of epicardial adipose tissue in cardiac biology: classic concepts and emerging roles.

Classic concepts about the role of epicardial adipose tissue (EpAT) in heart physiology include its role in cardiac metabolism, mechanical protection of coronaries, innervation and possibly cryoprotection of the heart too. Nevertheless, recent evidence has revealed that epicardial adipose tissue regulates multiple aspects of cardiac biology including myocardial redox state, intracellular Ca2+ cycling, the electrophysiological and contractile properties of cardiomyocytes, cardiac fibrosis as well as coronary atherosclerosis progression. Moreover, it is now understood that the communication between EpAT and the heart is regulated by complex bidirectional pathways, since not only do adipokines regulate cardiac function, but also the heart affects EpAT biology via paracrine 'reverse' signalling. Such complex interactions as well as epicardial fat accumulation as a consequence of cardiac disease and epicardium to adipocyte differentiation should be taken into account by the clinical studies investigating EpAT as a risk marker and its potential as a therapeutic target against cardiovascular disease. Further in-depth exploration of the molecular mechanisms regulating the cross-talk between the heart and EpAT is expected to enhance our understanding regarding the role of the latter in cardiac physiology and relevant disease mechanisms.

Beta-Adrenoceptor Stimulation Reveals Ca2+ Waves and Sarcoplasmic Reticulum Ca2+ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure.

Abnormal cellular Ca2+ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca2+ transient amplitude due to decreased sarcoplasmic reticulum Ca2+ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca2+ release events which occur when sarcoplasmic reticulum Ca2+ content exceeds a certain threshold level. Such Ca2+ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca2+ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca2+ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca2+ transients and Ca2+ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca2+ content, decreased Ca2+ transient amplitude, and increased frequency of Ca2+ waves. These results are in line with a decreased threshold for diastolic Ca2+ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca2+ release were increased THR286 phosphorylation of Ca2+/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca2+ content and increased propensity for diastolic Ca2+ release events in ventricular cardiomyocytes from rats with heart failure after myocardial infarction, and that these phenomena are also found in rats with large myocardial infarctions without heart failure development. Importantly, beta-adrenoceptor stimulation is necessary to reveal these perturbations in Ca2+ handling after a myocardial infarction.

CARD9 knockout ameliorates myocardial dysfunction associated with high fat diet-induced obesity

Obesity is associated with chronic inflammation which plays a critical role in the development of cardiovascular dysfunction. Because the adaptor protein caspase recruitment domain-containing protein 9 (CARD9) in macrophages regulates innate immune responses via activation of pro-inflammatory cytokines, we hypothesize that CARD9 mediates the pro-inflammatory signaling associated with obesity en route to myocardial dysfunction.C57BL/6 wild-type (WT) and CARD9−/− mice were fed normal diet (ND, 12% fat) or a high fat diet (HFD, 45% fat) for 5months. At the end of 5-month HFD feeding, cardiac function was evaluated using echocardiography. Cardiomyocytes were isolated and contractile properties were measured. Immunofluorescence was performed to detect macrophage infiltration in the heart. Heart tissue homogenates, plasma, and supernatants from isolated macrophages were collected to measure the concentrations of pro-inflammatory cytokines using ELISA kits. Western immunoblotting analyses were performed on heart tissue homogenates and isolated macrophages to explore the underlying signaling mechanism(s). CARD9 knockout alleviated HFD-induced insulin resistance and glucose intolerance, prevented myocardial dysfunction with preserved cardiac fractional shortening and cardiomyocyte contractile properties. CARD9 knockout also significantly decreased the number of infiltrated macrophages in the heart with reduced myocardium-, plasma-, and macrophage-derived cytokines including IL-6, IL-1β and TNFα. Finally, CARD9 knockout abrogated the increase of p38 MAPK phosphorylation, the decrease of LC3BII/LC3BI ratio and the up-regulation of p62 expression in the heart induced by HFD feeding and restored cardiac autophagy signaling.In conclusion, CARD9 knockout ameliorates myocardial dysfunction associated with HFD-induced obesity, potentially through reduction of macrophage infiltration, suppression of p38 MAPK phosphorylation, and preservation of autophagy in the heart.

Spatiotemporal control of cardiac anisotropy using dynamic nanotopographic cues

Coordinated extracellular matrix spatiotemporal reorganization helps regulate cellular differentiation, maturation, and function in vivo, and is therefore vital for the correct formation, maintenance, and healing of complex anatomic structures. In order to evaluate the potential for cultured cells to respond to dynamic changes in their in vitro microenvironment, as they do in vivo, the collective behavior of primary cardiac muscle cells cultured on nanofabricated substrates with controllable anisotropic topographies was studied. A thermally induced shape memory polymer (SMP) was employed to assess the effects of a 90° transition in substrate pattern orientation on the contractile direction and structural organization of cardiomyocyte sheets. Cardiomyocyte sheets cultured on SMPs exhibited anisotropic contractions before shape transition. 48 h after heat-induced shape transition, the direction of cardiomyocyte contraction reoriented significantly and exhibited a bimodal distribution, with peaks at ∼45 and −45° (P < 0.001). Immunocytochemical analysis highlighted the significant structural changes that the cells underwent in response to the shift in underlying topography. The presented results demonstrate that initial anisotropic nanotopographic cues do not permanently determine the organizational fate or contractile properties of cardiomyocytes in culture. Given the importance of surface cues in regulating primary and stem cell development, investigation of such tunable nanotopographies may have important implications for advancing cellular maturation and performance in vitro, as well as improving our understanding of cellular development in response to dynamic biophysical cues.

Overexpression myocardial inducible nitric oxide synthase exacerbates cardiac dysfunction and beta-adrenergic desensitization in experimental hypothyroidism

Altered nitric oxide synthase (NOS) has been implicated in the pathophysiology of heart failure (HF). Recent evidence links hypothyroidism to the pathology of HF. However, the precise mechanisms are incompletely understood. The alterations and functional effects of cardiac NOS in hypothyroidism are unknown. We tested the hypothesis that hypothyroidism increases cardiomyocyte inducible NOS (iNOS) expression, which plays an important role in hypothyroidism-induced depression of cardiomyocyte contractile properties, [Ca(2+)]i transient ([Ca(2+)]iT), and β-adrenergic hyporesponsiveness. We simultaneously evaluated LV functional performance and compared myocyte three NOS, β-adrenergic receptors (AR) and SERCA2a expressions and assessed cardiomyocyte contractile and [Ca(2+)]iT responses to β-AR stimulation with and without pretreatment of iNOS inhibitor (1400 W, 10(-5)mol/L) in 26 controls and 26 rats with hypothyroidism induced by methimazole (~30 mg/kg/day for 8 weeks in the drinking water). Compared with controls, in hypothyroidism, total serum T3 and T4 were significantly reduced followed by significantly decreased LV contractility (EES) with increased LV time constant of relaxation. These LV abnormalities were accompanied by concomitant significant decreases in myocyte contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca(2+)]iT. In hypothyroidism, isoproterenol (10(-8)M) produced significantly smaller increases in dL/dtmax, dR/dtmax and [Ca(2+)]iT. These changes were associated with decreased β1-AR and SERCA2a, but significantly increased iNOS. Moreover, only in hypothyroidism, pretreatment with iNOS inhibitor significantly improved basal and isoproterenol-stimulated myocyte contraction, relaxation and [Ca(2+)]iT. Hypothyroidism produces intrinsic defects of LV myocyte force-generating capacity and relaxation with β-AR desensitization. Up-regulation of cardiomyocyte iNOS may promote progressive cardiac dysfunction in hypothyroidism.

Cardiac-Specific Knockout of ETA Receptor Mitigates Paraquat-Induced Cardiac Contractile Dysfunction.

Paraquat (1,1'-dim ethyl-4-4'-bipyridinium dichloride), a highly toxic quaternary ammonium herbicide widely used in agriculture, exerts potent toxic prooxidant effects resulting in multi-organ failure including the lung and heart although the underlying mechanism remains elusive. Recent evidence suggests possible involvement of endothelin system in paraquat-induced acute lung injury. This study was designed to examine the role of endothelin receptor A (ETA) in paraquat-induced cardiac contractile and mitochondrial injury. Wild-type (WT) and cardiac-specific ETA receptor knockout mice were challenged to paraquat (45mg/kg, i.p.) for 48h prior to the assessment of echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties, as well as apoptosis and mitochondrial damage. Levels of the mitochondrial proteins for biogenesis and oxidative phosphorylation including UCP2, HSP90 and PGC1α were evaluated. Our results revealed that paraquat elicited cardiac enlargement, mechanical anomalies including compromised echocardiographic parameters (elevated left ventricular end-systolic and end-diastolic diameters as well as reduced factional shortening), suppressed cardiomyocyte contractile function, intracellular Ca(2+) handling, overt apoptosis and mitochondrial damage. ETA receptor knockout itself failed to affect myocardial function, apoptosis, mitochondrial integrity and mitochondrial protein expression. However, ETA receptor knockout ablated or significantly attenuated paraquat-induced cardiac contractile and intracellular Ca(2+) defect, apoptosis and mitochondrial damage. Taken together, these findings revealed that endothelin system in particular the ETA receptor may be involved in paraquat-induced toxic myocardial contractile anomalies possibly related to apoptosis and mitochondrial damage.

RETRACTED: Akt2 knockout alleviates prolonged caloric restriction-induced change in cardiac contractile function through regulation of autophagy

Caloric restriction leads to changes in heart geometry and function although the underlying mechanism remains elusive. Autophagy, a conserved pathway for degradation of intracellular proteins and organelles, preserves energy and nutrient in the face of caloric insufficiency. This study was designed to examine the role of Akt2 in prolonged caloric restriction-induced change in cardiac homeostasis and the underlying mechanism(s) involved. Wild-type (WT) and Akt2 knockout mice were calorie restricted (by 40%) for 30weeks. Echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties, autophagy and its regulatory proteins were evaluated. Caloric restriction compromised echocardiographic indices (decreased left ventricular mass, left ventricular diameters and cardiac output), cardiomyocyte contractile and intracellular Ca(2+) properties associated with dampened SERCA2a phosphorylation, upregulated phospholamban and autophagy (Beclin-1, Atg7, LC3BII-to-LC3BI ratio), increased autophagy adaptor protein p62, elevated phosphorylation of AMPK, Akt2 and the Akt downstream signal molecule TSC2, the effects of which with the exception of autophagy protein markers (Beclin-1, Atg7, LC3B) and AMPK were mitigated or significantly alleviated by Akt2 knockout. Lysosomal inhibition using bafilomycin A1 negated Akt2 knockout-induced protective effect on p62. Evaluation of downstream signaling molecules of Akt and AMPK including mTOR and ULK1 revealed that caloric restriction suppressed and promoted phosphorylation of mTOR and ULK1, respectively, without affecting total mTOR and ULK1 expression. Akt2 knockout significantly augmented caloric restriction-induced responses on mTOR and ULK1. Taken together, these data suggest a beneficial role of Akt2 knockout in preservation of cardiac homeostasis against prolonged caloric restriction-induced pathological changes possibly through facilitating autophagy. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy."

Abstract 314: The Interaction Of The Estrogen Receptor Alpha With The Cardiac Myosin Essential Light Chain Isoform 4 As A New Regulatory Mechanism In The Heart

Estrogen (17β-estradiol, E2) is one of the key regulators of growth, differentiation and physiological function in different tissues, including the heart. The effects of E2 are mainly mediated by estrogen receptor (ER) alpha and beta, which act in concert with many cofactors to mediate estrogenic effects. So far, only a few cofactors of ER have been described in the human heart. To gain a better understanding of E2-mediated ERα action in the human heart, we identified and characterized the novel interaction partners of ERα Yeast two hybrid screening of a human heart cDNA library revealed that ERα interacts with the cardiac myosin essential light chain isoform 4 (ALC1) in presence of E2. ALC1, as a member of contractile proteins, is expressed in the fetal heart and becomes restricted to the atria of the adult heart under physiological conditions, and is reexpressed in ventricle of adult hypertrophic hearts. This switch is accompanied by alteration of contractile performance, thus improving the heart function. Retransformation experiments showed that ALC1 interacts with full-length ERα and ERα-EF domain in presence of E2. The interaction of ERα with ALC1 was also confirmed by Co-IP in human atrium. Double irnmunofluorescence (IF) analysis of paraffin-embedded sections from human atrium tissues showed co-localization of ERα and ALC1 proteins in a striated sarcomeric pattern. Co-localization corresponds most likely to the H-zone of sarcomere. IF analysis of AC16 cells (a human cardiomyocytes cell line) showed that ERα and ALC1 were mainly localized in the cytoplasm in the absence of E2. However, E2 treatment of AC16 cells led to a translocation of ALC1 and ERα into the nuclei of AC16 cells, where they also co-localized. Expression analysis in AC16 cells showed that E2 increases the expression of ALC1 gene (MYL4). Using ERE-based luciferase reporter assays we showed that E2-induced interaction of ERα with the ALC1 represses the transcriptional activity of ERα. We characterize for the first time an E2-regulated interaction of ALC1 with ERα in cardiomyocytes that may be crucial in physiological and/or pathological processes by regulating of transcriptional activity of ERa in the heart and/or by modulating contractile properties of cardiomyocytes.

Exercise training prior to myocardial infarction attenuates cardiac deterioration and cardiomyocyte dysfunction in rats

OBJECTIVES:The present study was performed to investigate 1) whether aerobic exercise training prior to myocardial infarction would prevent cardiac dysfunction and structural deterioration and 2) whether the potential cardiac benefits of aerobic exercise training would be associated with preserved morphological and contractile properties of cardiomyocytes in post-infarct remodeled myocardium.METHODS:Male Wistar rats underwent an aerobic exercise training protocol for eight weeks. The rats were then assigned to sham surgery (SHAM), sedentary lifestyle and myocardial infarction or exercise training and myocardial infarction groups and were evaluated 15 days after the surgery. Left ventricular tissue was analyzed histologically, and the contractile function of isolated myocytes was measured. Student's t-test was used to analyze infarct size and ventricular wall thickness, and the other parameters were analyzed by the Kruskal-Wallis test followed by Dunn's test or a one-way analysis of variance followed by Tukey's test (p<0.05).RESULTS:Myocardial infarctions in exercise-trained animals resulted in a smaller myocardial infarction extension, a thicker infarcted wall and less collagen accumulation as compared to myocardial infarctions in sedentary animals. Myocardial infarction-induced left ventricular dilation and cardiac dysfunction, as evaluated by +dP/dt and -dP/dt, were both prevented by previous aerobic exercise training. Moreover, aerobic exercise training preserved cardiac myocyte shortening, improved the maximum shortening and relengthening velocities in infarcted hearts and enhanced responsiveness to calcium.CONCLUSION:Previous aerobic exercise training attenuated the cardiac dysfunction and structural deterioration promoted by myocardial infarction, and such benefits were associated with preserved cardiomyocyte morphological and contractile properties.

RETRACTED ARTICLE: Cardiomyocyte-specific deletion of endothelin receptor A rescues aging-associated cardiac hypertrophy and contractile dysfunction: role of autophagy

Cardiac aging is manifested as cardiac remodeling and contractile dysfunction although precise mechanisms remain elusive. This study was designed to examine the role of endothelin-1 (ET-1) in aging-associated myocardial morphological and contractile defects. Echocardiographic and cardiomyocyte contractile properties were evaluated in young (5-6months) and old (26-28months) C57BL/6 wild-type and cardiomyocyte-specific ET(A) receptor knockout (ETAKO) mice. Cardiac ROS production and histology were examined. Our data revealed that ETAKO mice displayed an improved survival. Aging increased plasma levels of ET-1 and Ang II, compromised cardiac function (fractional shortening, cardiomyocyte peak shortening, maximal velocity of shortening/relengthening and prolonged relengthening) and intracellular Ca(2+) handling (reduced intracellular Ca(2+) release and decay), the effects of which with the exception of ET-1 and Ang II levels was improved by ETAKO. Histological examination displayed cardiomyocyte hypertrophy and interstitial fibrosis associated with cardiac remodeling in aged C57 mice, which were alleviated in ETAKO mice. Aging promoted ROS generation, protein damage, ER stress, upregulated GATA4, ANP, NFATc3 and the autophagosome cargo protein p62, downregulated intracellular Ca(2+) regulatory proteins SERCA2a and phospholamban as well as the autophagic markers Beclin-1, Atg7, Atg5 and LC3BII, which were ablated by ETAKO. ET-1 triggered a decrease in autophagy and increased hypertrophic markers in vitro, the effect of which were reversed by the ET(A) receptor antagonist BQ123 and the autophagy inducer rapamycin. Antagonism of ET(A), but not ET(B) receptor, rescued cardiac aging, which was negated by autophagy inhibition. Taken together, our data suggest that cardiac ET(A) receptor ablation protects against aging-associated myocardial remodeling and contractile dysfunction possibly through autophagy regulation.