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The zebrafish (Danio rerio) is an increasingly popular model organism in cardiovascular research. Major insights into cardiac developmental processes have been gained by studies of embryonic zebrafish. However, the utility of zebrafish for modeling adult-onset heart disease has been limited by a lack of robust methods for in vivo evaluation of cardiac function. We established a physiological protocol for underwater zebrafish echocardiography using high frequency ultrasound, and evaluated its reliability in detecting altered cardiac function in two disease models. Serial assessment of cardiac function was performed in wild-type zebrafish aged 3 to 12 months and the effects of anesthetic agents, age, sex, and background strain were evaluated. There was a varying extent of bradycardia and ventricular contractile impairment with different anesthetic drugs and doses, with tricaine 0.75 mmolL−1 having a relatively more favorable profile. When compared with males, female fish were larger and had more measurement variability. Although age-related increments in ventricular chamber size were greater in females than males, there were no sex differences when data were normalized to body size. Systolic ventricular function was similar in both sexes at all time-points, but differences in diastolic function were evident from 6 months onwards. Wild-type fish of both sexes showed a reliance on atrial contraction for ventricular diastolic filling. Echocardiographic evaluation of adult zebrafish with diphtheria toxin-induced myocarditis or anemia-induced volume overload accurately identified ventricular dilation and altered contraction, with suites of B-mode, ventricular strain, pulsed-wave Doppler and tissue Doppler indices showing concordant changes indicative of myocardial hypocontractility or hypercontractility, respectively. Repeatability, intra-observer and inter-observer correlations for echocardiographic measurements were high. We demonstrate that high frequency echocardiography allows reliable in vivo cardiac assessment in adult zebrafish and make recommendations for optimizing data acquisition and analysis. This enabling technology reveals new insights into zebrafish cardiac physiology and provides an imaging platform for zebrafish-based translational research.

Pathological left ventricular hypertrophy (LVH) occurs in response to pressure overload and remains the single most important clinical predictor of cardiac mortality. The molecular pathways in the induction of pressure overload LVH are potential targets for therapeutic intervention. Current treatments aim to remove the pressure overload stimulus for LVH, but do not completely reverse adverse cardiac remodelling. Although numerous molecular signalling steps in the induction of LVH have been identified, the initial step by which mechanical stretch associated with cardiac pressure overload is converted into a chemical signal that initiates hypertrophic signalling remains unresolved. In this study, we show that selective deletion of transient receptor potential melastatin 4 (TRPM4) channels in mouse cardiomyocytes results in an approximately 50% reduction in the LVH induced by transverse aortic constriction. Our results suggest that TRPM4 channel is an important component of the mechanosensory signalling pathway that induces LVH in response to pressure overload and represents a potential novel therapeutic target for the prevention of pathological LVH.

Laminopathies are a group of disorders caused by mutations in the LMNA gene that encodes the nuclear lamina proteins, lamin A and lamin C; their pathophysiological basis is unknown. We report that lamin A/C-deficient (Lmna(-/-)) mice develop rapidly progressive dilated cardiomyopathy (DCM) characterized by left ventricular (LV) dilation and reduced systolic contraction. Isolated Lmna(-/-) myocytes show reduced shortening with normal baseline and peak amplitude of Ca(2+) transients. Lmna(-/-) LV myocyte nuclei have marked alterations of shape and size with central displacement and fragmentation of heterochromatin; these changes are present but less severe in left atrial nuclei. Electron microscopy of Lmna(-/-) cardiomyocytes shows disorganization and detachment of desmin filaments from the nuclear surface with progressive disruption of the cytoskeletal desmin network. Alterations in nuclear architecture are associated with defective nuclear function evidenced by decreased SREBP1 import, reduced PPARgamma expression, and a lack of hypertrophic gene activation. These findings suggest a model in which the primary pathophysiological mechanism in Lmna(-/-) mice is defective force transmission resulting from disruption of lamin interactions with the muscle-specific desmin network and loss of cytoskeletal tension. Despite severe DCM, defects in nuclear function prevent Lmna(-/-) cardiomyocytes from developing compensatory hypertrophy and accelerate disease progression.

Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like most plasma membrane ion channel proteins they must pass through biosynthetic quality control in the endoplasmic reticulum that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the ‘cap’ region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for N-glycosylation in the ‘propeller’ regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated Piezo1 protein. Thus the N-linked glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.Li et al. investigates the role of N-linked glycosylation for the function of mechanosensitive ion channel Piezo1. They show that disease-linked loss of function mutations in Piezo1 that are trafficking defective lack N-linked glycosylation.

BackgroundAccurate classification of aortic stenosis (AS) severity remains challenging despite detailed echocardiographic assessment. Adjudication of severity is informed by subjective interpretation of aortic leaflet motion from the first image parasternal long axis (PLAX) view, but quantitative metrics of leaflet motion currently do not exist. The objectives of the study were to echocardiographically quantify aortic leaflet motion using the PLAX view and correlate motion data with Doppler-derived hemodynamic indices of disease severity, and predict significant AS using these isolated motion data.MethodsPLAX loops from 200 patients with and without significant AS were analyzed. Linear and angular motion of the anterior (right coronary) leaflet were quantified and compared between severity grades. Three simple supervised machine learning classifiers were then trained to distinguish significant (moderate or worse) from nonsignificant AS and individual severity grades.ResultsLinear and angular displacement demonstrated strong correlation with aortic valve area (r = 0.81 and r = 0.74, respectively). Severe AS cases demonstrated global leaflet motion of 2.1 mm, compared with 3.6 mm for moderate cases (P < 0.01) and 9.2 mm for control cases (P < 0.01). Severe cases demonstrated mean global angular rotation of 11°, significantly less than moderate (18°, P < 0.01) and normal cases (47°, P < 0.01). Using these novel metrics, a simple supervised machine learning model predicted significant AS with an accuracy of 90% and area under the receiver operator characteristics curve (AUC) of 0.96. Prediction of individual severity class was achieved with an accuracy of 72.5% and AUC of 0.88.ConclusionsAdvancing severity of AS is associated with significantly reduced linear and angular leaflet displacement. Leaflet motion data can accurately classify AS using a single parasternal long axis view, without the need for hemodynamic or Doppler assessment. Our model, grounded in biological plausibility, simple linear algebra, and supervised machine learning, provides a highly explainable approach to disease identification and may hold significant clinical utility for the diagnosis and classification of AS.

A 58-year-old woman with metastatic breast cancer and Coombs-positive autoimmune haemolytic anaemia presented in April, 2014, after developing right arm weakness 8 hours into a long-haul flight. Spontaneous echocardiographic contrast is usually caused by rouleaux formation, most commonly associated with low blood velocity and shear rates in the left atrium with atrial fibrillation,1,2 associated with an increased risk of stroke.3 Fibrinogen-mediated and other plasma protein-mediated red cell aggregation is largely responsible for its appearance,4,5 but its presence in heart chambers with normal function should raise suspicion of a hypercoagulable state.

Gq-coupled receptors are thought to play a critical role in the induction of left ventricular hypertrophy (LVH) secondary to pressure overload, although mechano-sensitive channel activation by a variety of mechanisms has also been proposed, and the relative importance of calcineurin- and calmodulin kinase II (CaMKII)-dependent hypertrophic pathways remains controversial. To determine the mechanisms regulating the induction of LVH in response to mechanical pressure overload. Transgenic mice with cardiac-targeted inhibition of Gq-coupled receptors (GqI mice) and their non-transgenic littermates (NTL) were subjected to neurohumoral stimulation (continuous, subcutaneous angiotensin II (AngII) infusion for 14 days) or mechanical pressure overload (transverse aortic arch constriction (TAC) for 21 days) to induce LVH. Candidate signaling pathway activation was examined. As expected, LVH observed in NTL mice with AngII infusion was attenuated in heterozygous (GqI ) mice and absent in homozygous (GqI ) mice. In contrast, LVH due to TAC was unaltered by either heterozygous or homozygous Gq inhibition. Gene expression of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and α-skeletal actin (α-SA) was increased 48 h after AngII infusion or TAC in NTL mice; in GqI mice, the increases in ANP, BNP and α-SA in response to AngII were completely absent, as expected, but all three increased after TAC. Increased nuclear translocation of nuclear factor of activated T-cells c4 (NFATc4), indicating calcineurin pathway activation, occurred in NTL mice with AngII infusion but not TAC, and was prevented in GqI mice infused with AngII. Nuclear and cytoplasmic CaMKIIδ levels increased in both NTL and GqI mice after TAC but not AngII infusion, with increased cytoplasmic phospho- and total histone deacetylase 4 (HDAC4) and increased nuclear myocyte enhancer factor 2 (MEF2) levels. Cardiac Gq receptors and calcineurin activation are required for neurohumorally mediated LVH but not for LVH induced by mechanical pressure overload (TAC). Rather, TAC-induced LVH is associated with activation of the CaMKII-HDAC4-MEF2 pathway.

To determine the mechanisms by which the α1A-adrenergic receptor (AR) regulates cardiac contractility. We reported previously that transgenic mice with cardiac-restricted α1A-AR overexpression (α1A-TG) exhibit enhanced contractility but not hypertrophy, despite evidence implicating this Gαq/11-coupled receptor in hypertrophy. Contractility, calcium (Ca(2+)) kinetics and sensitivity, and contractile proteins were examined in cardiomyocytes, isolated hearts and skinned fibers from α1A-TG mice (170-fold overexpression) and their non-TG littermates (NTL) before and after α1A-AR agonist stimulation and blockade, angiotensin II (AngII), and Rho kinase (ROCK) inhibition. Hypercontractility without hypertrophy with α1A-AR overexpression is shown to result from increased intracellular Ca(2+) release in response to agonist, augmenting the systolic amplitude of the intracellular Ca(2+) concentration [Ca(2+)]i transient without changing resting [Ca(2+)]i. In the absence of agonist, however, α1A-AR overexpression reduced contractility despite unchanged [Ca(2+)]i. This hypocontractility is not due to heterologous desensitization: the contractile response to AngII, acting via its Gαq/11-coupled receptor, was unaltered. Rather, the hypocontractility is a pleiotropic signaling effect of the α1A-AR in the absence of agonist, inhibiting RhoA/ROCK activity, resulting in hypophosphorylation of both myosin phosphatase targeting subunit 1 (MYPT1) and cardiac myosin light chain 2 (cMLC2), reducing the Ca(2+) sensitivity of the contractile machinery: all these effects were rapidly reversed by selective α1A-AR blockade. Critically, ROCK inhibition in normal hearts of NTLs without α1A-AR overexpression caused hypophosphorylation of both MYPT1 and cMLC2, and rapidly reduced basal contractility. We report for the first time pleiotropic α1A-AR signaling and the physiological role of RhoA/ROCK signaling in maintaining contractility in the normal heart.

Mutations in the giant sarcomeric protein titin (TTN) are a major cause for inherited forms of dilated cardiomyopathy (DCM). We have previously developed a mouse model that imitates a TTN truncation mutation we found in a large pedigree with DCM. While heterozygous Ttn knock-in mice do not display signs of heart failure under sedentary conditions, they recapitulate the human phenotype when exposed to the pharmacological stressor angiotensin II or isoproterenol. In this study we investigated the effects of pressure overload by transverse aortic constriction (TAC) in heterozygous (Het) Ttn knock-in mice. Two weeks after TAC, Het mice developed marked impairment of left ventricular ejection fraction (p<0.05), while wild-type (WT) TAC mice did not. Het mice also trended toward increased ventricular end diastolic pressure and volume compared to WT littermates. We found an increase in histologically diffuse cardiac fibrosis in Het compared to WT in TAC mice. This study shows that a pattern of DCM can be induced by TAC-mediated pressure overload in a TTN-truncated mouse model. This model enlarges our arsenal of cardiac disease models, adding a valuable tool to understand cardiac pathophysiological remodeling processes and to develop therapeutic approaches to combat heart failure.

Background Over 100 mammalian G protein-coupled receptors are yet to be matched with endogenous ligands; these so-called orphans are prospective drug targets for the treatment of disease. GPR37L1 is one such orphan, abundant in the brain and detectable as mRNA in the heart and kidney. GPR37L1 ablation was reported to cause hypertension and left ventricular hypertrophy, and thus, we sought to further define the role of GPR37L1 in blood pressure homeostasis. Methods We investigated the cardiovascular effects of GPR37L1 using wild-type (GPR37L1 wt/wt ) and null (GPR37L1 KO/KO ) mice established on a C57BL/6J background, both under baseline conditions and during AngII infusion. We profiled GPR37L1 tissue expression, examining the endogenous receptor by immunoblotting and a β-galactosidase reporter mouse by immunohistochemistry. Results GPR37L1 protein was abundant in the brain but not detectable in the heart and kidney. We measured blood pressure in GPR37L1 wt/wt and GPR37L1 KO/KO mice and found that deletion of GPR37L1 causes a female-specific increase in systolic, diastolic, and mean arterial pressures. When challenged with short-term AngII infusion, only male GPR37L1 KO/KO mice developed exacerbated left ventricular hypertrophy and evidence of heart failure, while the female GPR37L1 KO/KO mice were protected from cardiac fibrosis. Conclusions Despite its absence in the heart and kidney, GPR37L1 regulates baseline blood pressure in female mice and is crucial for cardiovascular compensatory responses in males. The expression of GPR37L1 in the brain, yet absence from peripheral cardiovascular tissues, suggests this orphan receptor is a hitherto unknown contributor to central cardiovascular control.