Imaging of Existing and Newly Translated Proteins Elucidates Mechanisms of Sarcomere Turnover.

BACKGROUND: How the sarcomeric complex is continuously turned over in long-living cardiomyocytes is unclear. According to the prevailing model of sarcomere maintenance, sarcomeres are maintained by cytoplasmic soluble protein pools with free recycling between pools and sarcomeres. METHODS: We imaged and quantified the turnover of expressed and endogenous sarcomeric proteins, including the giant protein titin, in cardiomyocytes in culture and in vivo, at the single cell and at the single sarcomere level using pulse-chase labeling of Halo-tagged proteins with covalent ligands. RESULTS: We disprove the prevailing protein pool model and instead show an ordered mechanism in which only newly translated proteins enter the sarcomeric complex while older ones are removed and degraded. We also show that degradation is independent of protein age and that proteolytic extraction is a rate-limiting step in the turnover. We show that replacement of sarcomeric proteins occurs at a similar rate within cells and across the heart and is slower in adult cells. CONCLUSIONS: Our findings establish a unidirectional replacement model for cardiac sarcomeres subunit replacement and identify their turnover principles.

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 mislocalization 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 demonstrated 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.