Localized translation and sarcomere maintenance requires ribosomal protein SA in mice.

Cardiomyocyte sarcomeres contain localized ribosomes, but the factors responsible for their localization and the significance of localized translation are unknown. Using proximity labeling, we identified Ribosomal Protein SA (RPSA) as a Z-line protein. In cultured cardiomyocytes, the loss of RPSA led to impaired local protein translation and reduced sarcomere integrity. By employing CAS9 expressing mice along with adeno-associated viruses expressing CRE recombinase and single-guide RNAs targeting Rpsa, we knocked out Rpsa in vivo and observed mis-localization of ribosomes and diminished local translation. These genetic mosaic mice with Rpsa knockout in a subset of cardiomyocytes developed dilated cardiomyopathy, featuring atrophy of RPSA-deficient cardiomyocytes, compensatory hypertrophy of unaffected cardiomyocytes, left ventricular dilation, and impaired contractile function. We demonstrate that RPSA C-terminal domain is sufficient for localization to the Z-lines and that if the microtubule network is disrupted RPSA loses its sarcomeric localization. These findings highlight RPSA as a ribosomal factor essential for ribosome localization to the Z-line, facilitating local translation and sarcomere maintenance.

RNA binding protein IGF2BP2 expression is induced by stress in the heart and mediates dilated cardiomyopathy.

The IGF2BP family of RNA binding proteins consists of three paralogs that regulate intracellular RNA localization, RNA stability, and translational control. Although IGF2BP1 and 3 are oncofetal proteins, IGF2BP2 expression is maintained in many tissues, including the heart, into adulthood. IGF2BP2 is upregulated in cardiomyocytes during cardiac stress and remodeling and returns to normal levels in recovering hearts. We wondered whether IGF2BP2 might play an adaptive role during cardiac stress and recovery. Enhanced expression of an IGF2BP2 transgene in a conditional, inducible mouse line leads to dilated cardiomyopathy (DCM) and death within 3-4 weeks in newborn or adult hearts. Downregulation of the transgene after 2 weeks, however, rescues these mice, with complete recovery by 12 weeks. Hearts overexpressing IGF2BP2 downregulate sarcomeric and mitochondrial proteins and have fragmented mitochondria and elongated, thinner sarcomeres. IGF2BP2 is also upregulated in DCM or myocardial infarction patients. These results suggest that IGF2BP2 may be an attractive target for therapeutic intervention in cardiomyopathies.

Dual specific phosphatases (DUSPs) in cardiac hypertrophy and failure.

Pressure overload and other stress stimuli elicit a host of adaptive and maladaptive signaling cascades that eventually lead to cardiac hypertrophy and heart failure. Among those, the mitogen-activated protein kinase (MAPK) signaling pathway has been shown to play a prominent role. The dual specificity phosphatases (DUSPs), also known as MAPK specific phosphatases (MKPs), that can dephosphorylate the MAPKs and inactivate them are gaining increasing attention as potential drug targets. Here we try to review recent advancements in understanding the roles of the different DUSPs, and the pathways that they regulate in cardiac remodeling. We focus on the regulation of three main MAPK branches - the p38 kinases, the c-Jun-N-terminal kinases (JNKs) and the extracellular signal-regulated kinases (ERK) by various DUSPs and try to examine their roles.

Microtubules orchestrate local translation to enable cardiac growth.

Hypertension, exercise, and pregnancy are common triggers of cardiac remodeling, which occurs primarily through the hypertrophy of individual cardiomyocytes. During hypertrophy, stress-induced signal transduction increases cardiomyocyte transcription and translation, which promotes the addition of new contractile units through poorly understood mechanisms. The cardiomyocyte microtubule network is also implicated in hypertrophy, but via an unknown role. Here, we show that microtubules are indispensable for cardiac growth via spatiotemporal control of the translational machinery. We find that the microtubule motor Kinesin-1 distributes mRNAs and ribosomes along microtubule tracks to discrete domains within the cardiomyocyte. Upon hypertrophic stimulation, microtubules redistribute mRNAs and new protein synthesis to sites of growth at the cell periphery. If the microtubule network is disrupted, mRNAs and ribosomes collapse around the nucleus, which results in mislocalized protein synthesis, the rapid degradation of new proteins, and a failure of growth, despite normally increased translation rates. Together, these data indicate that mRNAs and ribosomes are actively transported to specific sites to facilitate local translation and assembly of contractile units, and suggest that properly localized translation - and not simply translation rate - is a critical determinant of cardiac hypertrophy. In this work, we find that microtubule based-transport is essential to couple augmented transcription and translation to productive cardiomyocyte growth during cardiac stress.

Towards standardization of echocardiography for the evaluation of left ventricular function in adult rodents: a position paper of the ESC Working Group on Myocardial Function.

Echocardiography is a reliable and reproducible method to assess non-invasively cardiac function in clinical and experimental research. Significant progress in the development of echocardiographic equipment and transducers has led to the successful translation of this methodology from humans to rodents, allowing for the scoring of disease severity and progression, testing of new drugs, and monitoring cardiac function in genetically modified or pharmacologically treated animals. However, as yet, there is no standardization in the procedure to acquire echocardiographic measurements in small animals. This position paper focuses on the appropriate acquisition and analysis of echocardiographic parameters in adult mice and rats, and provides reference values, representative images, and videos for the accurate and reproducible quantification of left ventricular function in healthy and pathological conditions.

Risk Factors for the Development of Functional Tricuspid Regurgitation and Their Population-Attributable Fractions.

OBJECTIVES: The objective of this study was to determine risk factors for progression to hemodynamically significant tricuspid regurgitation (TR) and the population burden attributable to these risk factors. BACKGROUND: Few data are available with regard to risk factors associated with the development of hemodynamically significant functional TR. METHODS: A total of 1,552 subjects were studied beginning with an index echocardiogram demonstrating trivial or mild TR. Risk factors for progression to moderate or severe TR were determined by using logistic regression and classification trees. Population attributable fractions were calculated for each risk factor. RESULTS: During a median follow-up time of 38 (interquartile range [IQR]: 26 to 63) months, 292 patients (18.8%) developed moderate/severe TR. Independent predictors of TR progression were age, female sex, heart failure, pacemaker electrode, atrial fibrillation (AF), and indicators of left heart disease, including left atrial (LA) enlargement, elevated pulmonary artery pressure (PAP), and left-sided valvular disease. Classification and regression tree analysis demonstrated that the strongest predictors of TR progression were PAP of ≥36 mm Hg, LA enlargement, age ≥60 years, and AF. In the absence of these 4 risk factors, progression to moderate or severe TR occurred in ∼3% of patients. Age (28.4%) and PAP (20.5%) carried the highest population-attributable fractions for TR progression. In patients with TR progression, there was a marked concomitant increase of incident cases of elevated PAP (40%); mitral and aortic valve intervention (12%); reductions in left ventricular ejection fraction (19%), and new AF (32%) (all p < 0.01). CONCLUSIONS: TR progression is determined mainly by markers of increased left-sided filling pressures (PAP and LA enlargement), AF, and age. At the population level, age and PAP are the most important contributors to the burden of significant TR. TR progression entails a marked parallel increase in the severity of left-sided heart disease.

A simple adeno-associated virus-based approach for the generation of cardiac genetic models in rats.

Background: Heart failure is a major health problem and progress in this field relies on better understanding of the mechanisms and development of novel therapeutics using animal models. The rat may be preferable to the mouse as a cardiovascular disease model due to its closer physiology to humans and due to its large size that facilitates surgical and monitoring procedures. However, unlike the mouse, genetic manipulation of the rat genome is challenging. Methods: Here we developed a simple, refined, and robust cardiac-specific rat transgenic model based on an adeno-associated virus (AAV) 9 containing a cardiac troponin T promoter. This model uses a single intraperitoneal injection of AAV and does not require special expertise or equipment. Results: We characterize the AAV dose required to achieve a high cardiac specific level of expression of a transgene in the rat heart using a single intraperitoneal injection to neonates. We show that at this AAV dose GFP expression does not result in hypertrophy, a change in cardiac function or other evidence for toxicity. Conclusions: The model shown here allows easy and fast transgenic based disease modeling of cardiovascular disease in the rat heart, and can also potentially be expanded to deliver Cas9 and gRNAs or to deliver small hairpin (sh)RNAs to also achieve gene knockouts and knockdown in the rat heart.

Lack of Increased Cardiovascular Risk due to Functional Tricuspid Regurgitation in Patients with Left-Sided Heart Disease.

BACKGROUND: Significant tricuspid regurgitation (TR) is associated with higher risk for adverse cardiovascular outcomes. Left-sided heart disease (LHD) is a potentially important confounder of this association because it is strongly linked to both TR and clinical outcome. METHODS: We studied 5,886 patients who were followed for a period of 10 years after the index echocardiographic examination. The relationship between TR severity and the end point of admission for heart failure or cardiovascular mortality was analyzed using competing risk analysis, Cox model, and propensity score matching. RESULTS: Higher TR grade was associated with markers of LHD including left ventricular systolic dysfunction, valvular heart disease ≥ moderate, left atrial enlargement, and pulmonary hypertension (all P < .001). There was a significant interaction between TR and the presence of LHD with regard to the end point of heart failure in the competing risks model (P = .01) and the combined end point of heart failure and cardiovascular mortality (P = .02). In both models, moderate/severe TR was associated with higher risk for heart failure (hazard ratio [HR] = 3.10; 95% CI, 1.41-6.84; P = .005) and the combined end point of heart failure or cardiovascular mortality (HR = 2.75; 95% CI, 1.33-5.63, P = .006) only in patients without LHD. Propensity score matching yielded 350 patient pairs, of which 88% had LHD. The HR for heart failure or cardiovascular mortality at 10 years was 0.78 (95% CI, 0.56-1.08; P = .14) in the moderate/severe TR group as compared with the trivial/mild TR. CONCLUSIONS: Moderate or severe functional TR portends an increased risk for heart failure and cardiovascular mortality only when isolated, without concomitant LHD.

Extracellular signal-regulated kinase (ERK) activation preserves cardiac function in pressure overload induced hypertrophy.

BACKGROUND: Chronic pressure overload and a variety of mediators induce concentric cardiac hypertrophy. When prolonged, cardiac hypertrophy culminates in decreased myocardial function and heart failure. Activation of the extracellular signal-regulated kinase (ERK) is consistently observed in animal models of hypertrophy and in human patients, but its role in the process is controversial. METHODS: We generated transgenic mouse lines with cardiomyocyte restricted overexpression of intrinsically active ERK1, which similar to the observations in hypertrophy is phosphorylated on both the TEY and the Thr207 motifs and is overexpressed at pathophysiological levels. RESULTS: The activated ERK1 transgenic mice developed a modest adaptive hypertrophy with increased contractile function and without fibrosis. Following induction of pressure-overload, where multiple pathways are stimulated, this activation did not further increase the degree of hypertrophy but protected the heart through a decrease in the degree of fibrosis and maintenance of ventricular contractile function. CONCLUSIONS: The ERK pathway acts to promote a compensated hypertrophic response, with enhanced contractile function and reduced fibrosis. The activation of this pathway may be a therapeutic strategy to preserve contractile function when the pressure overload cannot be easily alleviated. The inhibition of this pathway, which is increasingly being used for cancer therapy on the other hand, should be used with caution in the presence of pressure-overload.

Analysis of rat cardiac myocytes and fibroblasts identifies combinatorial enhancer organization and transcription factor families.

Cardiac fibroblasts play key roles in both health and disease. Their regulatory elements, transcription factors (TFs), and mechanisms of expression control have not been fully elucidated. We used a differential open chromatin approach, coupled with active enhancer mark, transcriptomic, and computational TFs binding analysis to map cell-type-specific active enhancers in cardiac fibroblasts and cardiomyocytes, and outline the TFs families that control them. This approach was validated by its ability to uncover the known cardiomyocyte TF biology in an unbiased manner, and was then applied to cardiac fibroblasts. We identified Tead, Sox9, Smad, Tcf, Meis, Rbpj, and Runx1 as the main cardiac fibroblasts TF families. Our analysis shows that in both cell types, distal enhancers, containing concentrated combinatorial clusters of multiple tissue expressed TFs recognition motifs, are combinatorically clustered around tissue specific genes. This model for tissue specific gene expression in the heart supports the general "billboard" model for enhancer organization.

Localization of transcripts, translation, and degradation for spatiotemporal sarcomere maintenance.

The mechanisms responsible for maintaining macromolecular protein complexes, with their proper localization and subunit stoichiometry, are incompletely understood. Here we studied the maintenance of the sarcomere, the basic contractile macromolecular complex of cardiomyocytes. We performed single-cell analysis of cardiomyocytes using imaging of mRNA and protein synthesis, and demonstrate that three distinct mechanisms are responsible for the maintenance of the sarcomere: mRNAs encoding for sarcomeric proteins are localized to the sarcomere, ribosomes are localized to the sarcomere with localized sarcomeric protein translation, and finally, a localized E3 ubiquitin ligase allow efficient degradation of excess unincorporated sarcomeric proteins. We show that these mechanisms are distinct, required, and work in unison, to ensure both spatial localization, and to overcome the large variability in transcription. Cardiomyocytes simultaneously maintain all their sarcomeres using localized translation and degradation processes where proteins are continuously and locally synthesized at high rates, and excess proteins are continuously degraded.

Longitudinal two-dimensional strain for the diagnosis of left ventricular segmental dysfunction in patients with acute myocardial infarction.

The objectives of this study were to assess whether 2-dimensional strain (2DS) can detect left ventricular (LV) segmental dysfunction and to compare the diagnostic accuracy of various 2DS parameters. Multiple segmental longitudinal 2DS parameters were measured in 54 patients with a first myocardial infarction and single vessel coronary artery disease (age: 56 ± 11 years, 74% men, LV ejection fraction: 47 ± 10%, left anterior descending artery occlusion in 63%) and 14 age-matched subjects. 2DS parameters were compared to visual assessment of segmental function by multiple observers. Using receiver-operating characteristics analysis, the area under the curve (AUC) for peak systolic strain in diagnosing segmental dysfunction (akinetic or hypokinetic LV segments) and for diagnosing akinetic segments was 0.85 (95% confidence interval 0.83-0.88) and 0.88 (0.85-0.90), respectively (all P values < 0.001). Other 2DS strain parameters had similar (peak strain, peak strain rate) or lower (post-systolic shortening, time-to-peak strain, diastolic 2DS parameters) AUC values. An absolute value of peak systolic strain <16.8% (25th percentile in normal subjects) had high sensitivity (0.89) and negative predictive values (0.88), but low specificity (0.55) and positive predictive values (0.59) for diagnosing segmental dysfunction. Similar findings were observed using a cutoff of <13.3% (absolute value of 10th percentile) for diagnosing akinetic segments. Diagnostic accuracy was significantly worse for segments in which visual segmental assessment was discordant between observers. In conclusion, 2DS can be used to diagnose segmental LV dysfunction with high sensitivity but limited specificity. The diagnostic limitation of 2DS is partially related to the visual echocardiographic definition of segmental abnormality.

Salt-inducible kinase induces cytoplasmic histone deacetylase 4 to promote vascular calcification.

A pathologic osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) promotes arterial calcifications, a process associated with significant morbidity and mortality. The molecular pathways promoting this pathology are not completely understood. We studied VSMCs, mouse aortic rings, and human aortic valves and showed here that histone deacetylase 4 (HDAC4) is upregulated early in the calcification process. Gain- and loss-of-function assays demonstrate that HDAC4 is a positive regulator driving this pathology. HDAC4 can shuttle between the nucleus and cytoplasm, but in VSMCs, the cytoplasmic rather than the nuclear activity of HDAC4 promotes calcification, and a nuclear-localized mutant of HDAC4 fails to promote calcification. The cytoplasmic location and function of HDAC4 is controlled by the activity of salt-inducible kinase (SIK). Pharmacologic inhibition of SIK sends HDAC4 to the nucleus and inhibits the calcification process in VSMCs, aortic rings, and in vivo In the cytoplasm, HDAC4 binds and its activity depends on the adaptor protein ENIGMA (Pdlim7) to promote vascular calcification. These results establish a cytoplasmic role for HDAC4 and identify HDAC4, SIK, and ENIGMA as mediators of vascular calcification.

Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair.

BACKGROUND: Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions. METHODS: We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM. RESULTS: EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to ß-adrenergic stimulation mediated via canonical ß1- and ß2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair. CONCLUSIONS: We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions.

Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination.

The "canonical" proteasomal degradation signal is a substrate-anchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established-in both human and yeast cells-a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes.

Extracellular signal-regulated kinases 1/2 as regulators of cardiac hypertrophy.

Cardiac hypertrophy results from increased mechanical load on the heart and through the actions of local and systemic neuro-humoral factors, cytokines and growth factors. These mechanical and neuroendocrine effectors act through stretch, G protein-coupled receptors and tyrosine kinases to induce the activation of a myriad of intracellular signaling pathways including the extracellular signal-regulated kinases 1/2 (ERK1/2). Since most stimuli that provoke myocardial hypertrophy also elicit an acute phosphorylation of the threonine-glutamate-tyrosine (TEY) motif within the activation loops of ERK1 and ERK2 kinases, resulting in their activation, ERKs have long been considered promotors of cardiac hypertrophy. Several mouse models were generated in order to directly understand the causal role of ERK1/2 activation in the heart. These models include direct manipulation of ERK1/2 such as overexpression, mutagenesis or knockout models, manipulations of upstream kinases such as MEK1 and manipulations of the phosphatases that dephosphorylate ERK1/2 such as DUSP6. The emerging understanding from these studies, as will be discussed here, is more complex than originally considered. While there is little doubt that ERK1/2 activation or the lack of it modulates the hypertrophic process or the type of hypertrophy that develops, it appears that not all ERK1/2 activation events are the same. While much has been learned, some questions remain regarding the exact role of ERK1/2 in the heart, the upstream events that result in ERK1/2 activation and the downstream effector in hypertrophy.

Histone deacetylases as therapeutic targets--from cancer to cardiac disease.

Heart failure is a major public health problem in western society. Recently, agents that inhibit histone deacetylase (HDAC) enzymes were developed and approved by the FDA as anticancer agents. This breakthrough has provided the motivation to develop more potent and more selective HDAC inhibitors and to target other pathologic conditions with these drugs. Here we review experimental evidence showing that these drugs may be beneficial in preventing cardiac hypertrophy and heart failure. Several lines of evidence show that inhibitors of Class I HDACs can blunt cardiac hypertrophy and preserve cardiac function in several small animal models. In contrast, Class IIa HDACs appear to be suppressors of hypertrophy, though experimental data with small molecule blockers of this class is largely lacking. The effects of HDAC inhibition in cardiac diseases, the cell population in the heart that is targeted by HDAC blockers, as well as the relative roles of specific HDACs are still under intense investigation.

Adult cardiac expression of the activating transcription factor 3, ATF3, promotes ventricular hypertrophy.

Cardiac hypertrophy is an adaptive response to various mechanophysical and pathophysiological stresses. However, when chronic stress is sustained, the beneficial response turns into a maladaptive process that eventually leads to heart failure. Although major advances in the treatment of patients have reduced mortality, there is a dire need for novel treatments for cardiac hypertrophy. Accordingly, considerable efforts are being directed towards developing mice models and understanding the processes that lead to cardiac hypertrophy. A case in point is ATF3, an immediate early transcription factor whose expression is induced in various cardiac stress models but has been reported to have conflicting functional significance in hypertrophy. To address this issue, we generated a transgenic mouse line with tetracycline-regulated ATF3 cardiac expression. These mice allowed us to study the consequence of ATF3 expression in the embryo or during the adult period, thus distinguishing the effect of ATF3 on development versus pathogenesis of cardiac dysfunction. Importantly, ATF3 expression in adult mice resulted in rapid ventricles hypertrophy, heart dysfunction, and fibrosis. When combined with a phenylephrine-infusion pressure overload model, the ATF3 expressing mice displayed a severe outcome and heart dysfunction. In a complementary approach, ATF3 KO mice displayed a lower level of heart hypertrophy in the same pressure overload model. In summary, ectopic expression of ATF3 is sufficient to promote cardiac hypertrophy and exacerbates the deleterious effect of chronic pressure overload; conversely, ATF3 deletion protects the heart. Therefore, ATF3 may serve as an important drug target to reduce the detrimental consequences of heart hypertrophy.

Correction: Adult Cardiac Expression of the Activating Transcription Factor 3, ATF3, Promotes Ventricular Hypertrophy.

[This corrects the article DOI: 10.1371/journal.pone.0068396.].