BBA - Molecular Cell Research (v.1833, #4)

7th Ascona International Workshop on Cardiomyocyte Biology by Wolfgang A. Linke; Marcus C. Schaub (781-782).

Evolution and development of the building plan of the vertebrate heart by Bjarke Jensen; Tobias Wang; Vincent M. Christoffels; Antoon F.M. Moorman (783-794).
Early cardiac development involves the formation of a heart tube, looping of the tube and formation of chambers. These processes are highly similar among all vertebrates, which suggest the existence of evolutionary conservation of the building plan of the heart. From the jawless lampreys to man, T-box transcription factors like Tbx5 and Tbx20 are fundamental for heart formation, whereas Tbx2 and Tbx3 repress chamber formation on the sinu-atrial and atrioventricular borders. Also, electrocardiograms from different vertebrates are alike, even though the fish heart only has two chambers whereas the mammalian heart has four chambers divided by septa and in addition has much higher heart rates. We conclude that most features of the high-performance hearts of mammals and birds can be traced back to less developed traits in the hearts of ectothermic vertebrates. This article is part of a Special Issue entitled: Cardiomyocyte biology: Cardiac pathways of differentiation, metabolism and contraction.► The building plan to the vertebrate heart is remarkably well conserved in evolution. ► The molecular patterning of the heart imposes the electrical patterning. ► Transcription factors like Tbx5 and Tbx20 are crucial for heart formation. ► Tbx2 and Tbx3 repress chamber formation in border regions. ► Tbx2 and Tbx3 delineates the cardiac conduction system.
Keywords: Heart; Conduction system; Evolution; Development; Electrophysiology; Gene expression;

Second heart field cardiac progenitor cells in the early mouse embryo by Alexandre Francou; Edouard Saint-Michel; Karim Mesbah; Magali Théveniau-Ruissy; M. Sameer Rana; Vincent M. Christoffels; Robert G. Kelly (795-798).
At the end of the first week of mouse gestation, cardiomyocyte differentiation initiates in the cardiac crescent to give rise to the linear heart tube. The heart tube subsequently elongates by addition of cardiac progenitor cells from adjacent pharyngeal mesoderm to the growing arterial and venous poles. These progenitor cells, termed the second heart field, originate in splanchnic mesoderm medial to cells of the cardiac crescent and are patterned into anterior and posterior domains adjacent to the arterial and venous poles of the heart, respectively. Perturbation of second heart field cell deployment results in a spectrum of congenital heart anomalies including conotruncal and atrial septal defects seen in human patients. Here, we briefly review current knowledge of how the properties of second heart field cells are controlled by a network of transcriptional regulators and intercellular signaling pathways. Focus will be on 1) the regulation of cardiac progenitor cell proliferation in pharyngeal mesoderm, 2) the control of progressive progenitor cell differentiation and 3) the patterning of cardiac progenitor cells in the dorsal pericardial wall. Coordination of these three processes in the early embryo drives progressive heart tube elongation during cardiac morphogenesis. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► The heart tube elongates by addition of second heart field (SHF) progenitor cells. ► Perturbation of this process results in a spectrum of congenital heart anomalies. ► The regulation of proliferation, differentiation and patterning in the SHF are reviewed. ► Coordination of these processes drives progressive heart tube elongation.
Keywords: Heart development; Cardiac progenitor cell; Outflow tract;

Challenges measuring cardiomyocyte renewal by Mark H. Soonpaa; Michael Rubart; Loren J. Field (799-803).
Interventions to effect therapeutic cardiomyocyte renewal have received considerable interest of late. Such interventions, if successful, could give rise to myocardial regeneration in diseased hearts. Regenerative interventions fall into two broad categories, namely approaches based on promoting renewal of pre-existing cardiomyocytes and approaches based on cardiomyogenic stem cell activity. The latter category can be further subdivided into approaches promoting differentiation of endogenous cardiomyogenic stem cells, approaches wherein cardiomyogenic stem cells are harvested, amplified or enriched ex vivo, and subsequently engrafted into the heart, and approaches wherein an exogenous stem cell is induced to differentiate in vitro, and the resulting cardiomyocytes are engrafted into the heart. There is disagreement in the literature regarding the degree to which cardiomyocyte renewal occurs in the normal and injured heart, the mechanism(s) by which this occurs, and the degree to which therapeutic interventions can enhance regenerative growth. This review discusses several caveats which are encountered when attempting to measure cardiomyocyte renewal in vivo which likely contribute, at least in part, to the disagreement regarding the levels at which this occurs in normal, injured and treated hearts. This article is part of a Special Issue entitled: Cardiomyocyte biology: Cardiac pathways of differentiation, metabolism and contraction.► It is well accepted that cardiomyocyte renewal occurs in the adult mammalian heart. ► The level of cardiomyocyte renewal is subject to debate. ► The mechanism of cardiomyocyte renewal is subject to debate. ► Issues contributing to these differences are discussed.
Keywords: Cardiac regeneration; Cardiomyogenesis; Cardiomyocyte proliferation;

The magnitude of length dependent activation in striated muscle has been shown to vary with titin isoform. Recently, a rat that harbors a homozygous autosomal mutation (HM) causing preferential expression of a longer, giant titin isoform was discovered (Greaser et al. 2005). Here, we investigated the impact of titin isoform on myofilament force development and cross-bridge cycling kinetics as function of sarcomere length (SL) in tibialis anterior skeletal muscle isolated from wild type (WT) and HM. Skeletal muscle bundles from HM rats exhibited reductions in passive tension, maximal force development, myofilament calcium sensitivity, maximal ATP consumption, and tension cost at both short and long sarcomere length (SL = 2.8 μm and SL = 3.2 μm, respectively). Moreover, the SL-dependent changes in these parameters were attenuated in HM muscles. Additionally, myofilament Ca2 + activation–relaxation properties were assessed in single isolated myofibrils. Both the rate of tension generation upon Ca2 + activation (k ACT) as well as the rate of tension redevelopment following a length perturbation (k TR) were reduced in HM myofibrils compared to WT, while relaxation kinetics were not affected. We conclude that presence of a long isoform of titin in the striated muscle sarcomere is associated with reduced myofilament force development and cross-bridge cycling kinetics, and a blunting of myofilament length dependent activation. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► Myofilament length dependent activation (LDA) underlies the Starling Law of the heart. ► The giant molecule titin may play a pivotal role in length dependency. ► We studied striated muscle from mutant and wild-type rats with varied titin lengths. ► Long titin was associated with altered LDA. ► Conclusion, titin mediates the length signal so as to modulate LDA.
Keywords: Titin; Myofilament length dependent activation; Sarcomere length; Actin–myosin interaction;

Lysine methyltransferase Smyd2 regulates Hsp90-mediated protection of the sarcomeric titin springs and cardiac function by Tobias Voelkel; Christian Andresen; Andreas Unger; Steffen Just; Wolfgang Rottbauer; Wolfgang A. Linke (812-822).
Protein lysine methylation controls gene expression and repair of deoxyribonucleic acid in the nucleus but also occurs in the cytoplasm, where the role of this posttranslational modification is less understood. Members of the Smyd protein family of lysine methyltransferases are particularly abundant in the cytoplasm, with Smyd1 and Smyd2 being most highly expressed in the heart and in skeletal muscles. Smyd1 is a crucial myogenic regulator with histone methyltransferase activity but also associates with myosin, which promotes sarcomere assembly. Smyd2 methylates histones and non-histone proteins, such as the tumor suppressors, p53 and retinoblastoma protein, RB. Smyd2 has an intriguing function in the cytoplasm of skeletal myocytes, where it methylates the chaperone Hsp90, thus promoting the interaction of a Smyd2–methyl-Hsp90 complex with the N2A-domain of titin. This complex protects the sarcomeric I-band region and myocyte organization. We briefly summarize some novel functions of Smyd family members, with a focus on Smyd2, and highlight their role in striated muscles and cytoplasmic actions. We then provide experimental evidence that Smyd2 is also important for cardiac function. In the cytoplasm of cardiomyocytes, Smyd2 was found to associate with the sarcomeric I-band region at the titin N2A-domain. Binding to N2A occurred in vitro and in yeast via N-terminal and extreme C-terminal regions of Smyd2. Smyd2-knockdown in zebrafish using an antisense oligonucleotide morpholino approach strongly impaired cardiac performance. We conclude that Smyd2 and presumably several other Smyd family members are lysine methyltransferases which have, next to their nuclear activity, specific regulatory functions in the cytoplasm of heart and skeletal muscle cells. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► Smyd lysine methyltransferases have nuclear and cytoplasmic functions in the muscle. ► Smyd2 is enriched in myocytes and monomethylates cytoplasmic Hsp90 at K616. ► Smyd2–methylHsp90 interacts with and protects the titin N2-A domain. ► Smyd2–methyl-Hsp90–N2A-titin complex protects myocyte organization and function. ► Methyl-Hsp90 contributes to protein quality-control mechanisms in the heart and muscle.
Keywords: Protein quality control; Sarcomere; Titin; Hsp90; Protein methylation;

New insights into the functional significance of the acidic region of the unique N-terminal extension of cardiac troponin I by Marcus Henze; Stacey E. Patrick; Aaron Hinken; Sarah B. Scruggs; Paul Goldspink; Pieter P. de Tombe; Minae Kobayashi; Peipei Ping; Tomoyoshi Kobayashi; R. John Solaro (823-832).
Previous structural studies indicated a special functional role for an acidic region composed of residues 1–10 in the unique N-terminal peptide of cardiac troponin I (cTnI). Employing LC–MS/MS, we determined the presence of phosphorylation sites at S5/S6 in cTnI from wild type mouse hearts as well as in hearts of mice chronically expressing active protein kinase C-ε (PKCε) and exhibiting severe dilated cardiomyopathy (DCM). To determine the functional significance of these phosphorylations, we cloned and expressed wild-type cTnI, (Wt), and cTnI variants expressing pseudo-phosphorylation cTnI-(S5D), cTnI(S6D), as well as cTnI(S5A) and cTnI(S6A). We exchanged native Tn of detergent-extracted (skinned) fiber bundles with Tn reconstituted with the variant cTnIs and measured tension and cross-bridge dynamics. Compared to controls, myofilaments controlled by cTnI with pseudo-phosphorylation (S6D) or Ala substitution (S6A) demonstrated a significant depression in maximum tension, ATPase rate, and ktr, but no change in half-maximally activating Ca2 +. In contrast, pseudo-phosphorylation at position 5 (S5D) had no effects, although S5A induced an increase in Ca2 +-sensitivity with no change in maximum tension or ktr. We further tested the impact of acidic domain modifications on myofilament function in studies examining the effects of cTnI(A2V), a mutation linked to DCM. This mutation significantly altered the inhibitory activity of cTnI as well as cooperativity of activation of myofilament tension, but not when S23/S24 were pseudo-phosphorylated. Our data indicate a new functional and pathological role of amino acid modifications in the N-terminal acidic domain of cTnI that is modified by phosphorylations at cTnI(S23/S24). This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► N-terminus of cardiac troponin I interacts intra-molecularly with regulatory domains. ► We discovered novel phosphorylation sites in the acidic domain. ► Conformation and charge changes affected myofilament Ca-response. ► A cardiomyopathy linked mutation of Ala-2 to Val affects myofilament activation.
Keywords: Cardiac; Ca-activation; Phosphorylation; Cardiomyopathy; Thin filament;

A novel alpha-tropomyosin mutation associates with dilated and non-compaction cardiomyopathy and diminishes actin binding by Judith B.A. van de Meerakker; Imke Christiaans; Phil Barnett; Ronald H. Lekanne Deprez; Aho Ilgun; Olaf R.F. Mook; Marcel M.A.M. Mannens; Jan Lam; Arthur A.M. Wilde; Antoon F.M. Moorman; Alex V. Postma (833-839).
Background: Dilated cardiomyopathy (DCM) is characterized by idiopathic dilatation and systolic contractile dysfunction of the ventricle(s) leading to an impaired systolic function. The origin of DCM is heterogeneous, but genetic transmission of the disease accounts for up to 50% of the cases. Mutations in alpha-tropomyosin (TPM1), a thin filament protein involved in structural and regulatory roles in muscle cells, are associated with hypertrophic cardiomyopathy (HCM) and very rarely with DCM. Methods and results: Here we present a large four-generation family in which DCM is inherited as an autosomal dominant trait. Six family members have a cardiomyopathy with the age of diagnosis ranging from 5 months to 52 years. The youngest affected was diagnosed with dilated and non-compaction cardiomyopathy (NCCM) and died at the age of five. Three additional children died young of suspected heart problems. We mapped the phenotype to chromosome 15 and subsequently identified a missense mutation in TPM1, resulting in a p.D84N amino acid substitution. In addition we sequenced 23 HCM/DCM genes using next generation sequencing. The TPM1 p.D84N was the only mutation identified. The mutation co-segregates with all clinically affected family members and significantly weakens the binding of tropomyosin to actin by 25%. Conclusions: We show that a mutation in TPM1 is associated with DCM and a lethal, early onset form of NCCM, probably as a result of diminished actin binding caused by weakened charge–charge interactions. Consequently, the screening of TPM1 in patients and families with DCM and/or (severe, early onset forms of) NCCM is warranted. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► Large four-generation family, with dilated and non-compaction cardiomyopathy ► Phenotype mapped to chromosome 15, missense mutation in alpha-tropomyosin identified ► The mutation significantly reduces the binding of tropomyosin to actin by 25% ► TPM1 mutations can be associated with both DCM and a lethal, early onset form of NCCM
Keywords: Cardiomyopathy; Genetics; Myocardial contraction; Myosin;

The heart is an omnivore organ that requires constant energy production to match its functional demands. In the adult heart, adenosine‐5′‐triphosphate (ATP) production occurs mainly through mitochondrial fatty acid and glucose oxidation. The heart must constantly adapt its energy production in response to changes in substrate supply and work demands across diverse physiologic and pathophysiologic conditions. The cardiac myocyte maintains a high level of mitochondrial ATP production through a complex transcriptional regulatory network that is orchestrated by the members of the peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) family. There is increasing evidence that during the development of cardiac hypertrophy and in the failing heart, the activity of this network, including PGC-1, is altered. This review summarizes our current understanding of the perturbations in the gene regulatory pathways that occur during the development of heart failure. An appreciation of the role this regulatory circuitry serves in the regulation of cardiac energy metabolism may unveil novel therapeutic targets aimed at the metabolic disturbances that presage heart failure. This article is part of a Special Issue entitled:Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► Cardiac hypertrophy and failure are associated with changes in energy substrate utilization. ► ERR and PPAR play key roles in the regulation of cardiac fuel metabolism. ► PGC-1 integrates multiple stimuli to control cardiac myocyte energy metabolism.
Keywords: Heart failure; Mitochondria; Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α); Fatty acid oxidation (FAO);

Regulation and dysregulation of glucose transport in cardiomyocytes by Christophe Montessuit; René Lerch (848-856).
The ability of the heart muscle to derive energy from a wide variety of substrates provides the myocardium with remarkable capacity to adapt to the ever-changing metabolic environment depending on factors including nutritional state and physical activity. There is increasing evidence that loss of metabolic flexibility of the myocardium contributes to cardiac dysfunction in disease conditions such as diabetes, ischemic heart disease and heart failure. At the level of glucose metabolism reduced metabolic adaptation in most cases is characterized by impaired stimulation of transarcolemmal glucose transport in the cardiomyocytes in response to insulin, referred to as insulin resistance, or to other stimuli such as energy deficiency. This review discusses cellular mechanisms involved in the regulation of glucose uptake in cardiomyocytes and their potential implication in impairment of stimulation of glucose transport under disease conditions. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► Glucose metabolism is important for cardiomyocytes to cope with situations of stress. ► Glucose transport into cardiomyocytes occurs mostly through GLUT4 transporters. ► Translocation of GLUT4 regulates glucose transport into cardiomyocytes. ► Insulin and metabolic stress stimulate glucose transport by distinct mechanisms. ► Stimulation of glucose transport is impaired in the diabetic myocardium.
Keywords: Glucose transport; Cardiomyocyte; Insulin; Metabolic stress; AMPK; Diabetes;

Heart failure is a major cause of morbidity and mortality in the world. Cardiac energy metabolism, specifically fatty acid and glucose metabolism, is altered in heart failure and has been implicated as a contributing factor in the impaired heart function observed in heart failure patients. There is emerging evidence demonstrating that correcting these changes in energy metabolism by modulating mitochondrial oxidative metabolism may be an effective treatment for heart failure. Promising strategies include the downregulation of fatty acid oxidation and an increased coupling of glycolysis to glucose oxidation. Carnitine palmitoyl transferase I (CPT1), fatty acid β-oxidation enzymes, and pyruvate dehydrogenase kinase (PDK) are examples of metabolic targets for the treatment of heart failure. While targeting mitochondrial oxidative metabolism is a promising strategy to treat heart failure, further studies are needed to confirm the potential beneficial effect of modulating these metabolic targets as an approach to treating heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► Significant alterations in energy metabolism occur in the failing heart. ► Cardiac mitochondrial oxidative metabolism decreases in heart failure. ► Glycolysis increases in the failing heart. ► Optimizing energy metabolism is a novel approach to treat heart failure. ► Inhibiting fatty acid oxidation and stimulating glucose oxidation improves cardiac efficiency and function in heart failure.
Keywords: Heart failure; Glycolysis; Mitochondria; Fatty acid oxidation; Glucose oxidation; Carnitine palmitoyltransferase 1;

Posttranslational modifications of cardiac ryanodine receptors: Ca2 + signaling and EC-coupling by Ernst Niggli; Nina D. Ullrich; Daniel Gutierrez; Sergii Kyrychenko; Eva Poláková; Natalia Shirokova (866-875).
In cardiac muscle, a number of posttranslational protein modifications can alter the function of the Ca2 + release channel of the sarcoplasmic reticulum (SR), also known as the ryanodine receptor (RyR). During every heartbeat RyRs are activated by the Ca2 +-induced Ca2 + release mechanism and contribute a large fraction of the Ca2 + required for contraction. Some of the posttranslational modifications of the RyR are known to affect its gating and Ca2 + sensitivity. Presently, research in a number of laboratories is focused on RyR phosphorylation, both by PKA and CaMKII, or on RyR modifications caused by reactive oxygen and nitrogen species (ROS/RNS). Both classes of posttranslational modifications are thought to play important roles in the physiological regulation of channel activity, but are also known to provoke abnormal alterations during various diseases. Only recently it was realized that several types of posttranslational modifications are tightly connected and form synergistic (or antagonistic) feed-back loops resulting in additive and potentially detrimental downstream effects. This review summarizes recent findings on such posttranslational modifications, attempts to bridge molecular with cellular findings, and opens a perspective for future work trying to understand the ramifications of crosstalk in these multiple signaling pathways. Clarifying these complex interactions will be important in the development of novel therapeutic approaches, since this may form the foundation for the implementation of multi-pronged treatment regimes in the future. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► Cardiac ryanodine receptor (RyR) function is affected by posttranslational modifications. ► These modifications of the RyRs contribute to cardiac regulation and disease. ► RyR phosphorylation and oxidative/nitrosative modifications are relevant modifications. ► Various pathways of signaling crosstalk, antagonism and synergies are involved.
Keywords: Cardiac muscle; Ryanodine receptor; Calcium signaling; Oxidation; Nitrosation; Heart failure;

Multilayered regulation of cardiac ion channels by Shan-Shan Zhang; Robin M. Shaw (876-885).
Essential to beat-to-beat heart function is the ability for cardiomyocytes to propagate electrical excitation and generate contractile force. Both excitation and contractility depend on specific ventricular ion channels, which include the L-type calcium channel (LTCC) and the connexin 43 (Cx43) gap junction. Each of these two channels is localized to a distinct subdomain of the cardiomyocyte plasma membrane. In this review, we focus on regulatory mechanisms that govern the lifecycles of LTCC and Cx43, from their biogenesis in the nucleus to directed delivery to T-tubules and intercalated discs, respectively. We discuss recent findings on how alternative promoter usage, tissue-specific transcription, and alternative splicing determine precise ion channel expression levels within a cardiomyocyte. Moreover, recent work on microtubule and actin-dependent trafficking for Cx43 and LTCC are introduced. Lastly, we discuss how human cardiac disease phenotypes can be attributed to defects in distinct mechanisms of channel regulation at the level of gene expression and channel trafficking. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► Cx43 and Cav1.2 are essential for cardiac excitation and contraction. ► Transcriptional and alternative splicing mechanisms regulate cardiac ion channel expression. ► The cytoskeletal machinery can target cardiac ion channels to specific membrane subdomains. ► Altered ion channel expression and trafficking contribute to heart failure and arrhythmias.
Keywords: Connexin 43; Calcium channel; Directed targeting; Cytoskeleton; Gene expression;

The cardiac sodium current (I Na) is responsible for the rapid depolarization of cardiac cells, thus allowing for their contraction. It is also involved in regulating the duration of the cardiac action potential (AP) and propagation of the impulse throughout the myocardium. Cardiac I Na is generated by the voltage-gated Na+ channel, NaV1.5, a 2016-residue protein which forms the pore of the channel. Over the past years, hundreds of mutations in SCN5A, the human gene coding for NaV1.5, have been linked to many cardiac electrical disorders, including the congenital and acquired long QT syndrome, Brugada syndrome, conduction slowing, sick sinus syndrome, atrial fibrillation, and dilated cardiomyopathy. Similar to many membrane proteins, NaV1.5 has been found to be regulated by several interacting proteins. In some cases, these different proteins, which reside in distinct membrane compartments (i.e. lateral membrane vs. intercalated disks), have been shown to interact with the same regulatory domain of NaV1.5, thus suggesting that several pools of NaV1.5 channels may co-exist in cardiac cells. The aim of this review article is to summarize the recent works that demonstrate its interaction with regulatory proteins and illustrate the model that the sodium channel NaV1.5 resides in distinct and different pools in cardiac cells. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► NaV1.5, the cardiac sodium channel, is involved in channelopathies. ► NaV1.5 forms multi-protein macromolecular complexes. ► At least two distinct pools of NaV1.5 are found in cardiac cells.
Keywords: Sodium channel; Cardiac cells; Ion channels; Intercalated disk;

Calcium mishandling in diastolic dysfunction: Mechanisms and potential therapies by Michelle L. Asp; Joshua J. Martindale; Frazer I. Heinis; Wang Wang; Joseph M. Metzger (895-900).
Diastolic dysfunction is characterized by slow or incomplete relaxation of the ventricles during diastole, and is an important contributor to heart failure pathophysiology. Clinical symptoms include fatigue, shortness of breath, and pulmonary and peripheral edema, all contributing to decreased quality of life and poor prognosis. There are currently no therapies available that directly target the heart pump defects in diastolic function. Calcium mishandling is a hallmark of heart disease and has been the subject of a large body of research. Efforts are ongoing in a number of gene therapy approaches to normalize the function of calcium handling proteins such as sarcoplasmic reticulum calcium ATPase. An alternative approach to address calcium mishandling in diastolic dysfunction is to introduce calcium buffers to facilitate relaxation of the heart. Parvalbumin is a calcium binding protein found in fast-twitch skeletal muscle and not normally expressed in the heart. Gene transfer of parvalbumin into normal and diseased cardiac myocytes increases relaxation rate but also markedly decreases contraction amplitude. Although parvalbumin binds calcium in a delayed manner, it is not delayed enough to preserve full contractility. Factors contributing to the temporal nature of calcium buffering by parvalbumin are discussed in relation to remediation of diastolic dysfunction. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► Parvalbumin, a calcium buffer, facilitates rapid relaxation in fast-twitch muscle. ► Viral gene transfer of parvalbumin in cardiac myocytes hastens relaxation. ► Calcium buffering represents a potential gene therapeutic strategy for diastolic heart failure.
Keywords: Diastolic dysfunction; Heart failure; Parvalbumin; Gene therapy; Calcium;

A-kinase anchoring proteins: Molecular regulators of the cardiac stress response by Dario Diviani; Darko Maric; Irene Pérez López; Sabrina Cavin; Cosmo D. del Vescovo (901-908).
In response to stress or injury the heart undergoes a pathological remodeling process, associated with hypertrophy, cardiomyocyte death and fibrosis, that ultimately causes cardiac dysfunction and heart failure. It has become increasingly clear that signaling events associated with these pathological cardiac remodeling events are regulated by scaffolding and anchoring proteins, which allow coordination of pathological signals in space and time. A-kinase anchoring proteins (AKAPs) constitute a family of functionally related proteins that organize multiprotein signaling complexes that tether the cAMP-dependent protein kinase (PKA) as well as other signaling enzymes to ensure integration and processing of multiple signaling pathways. This review will discuss the role of AKAPs in the cardiac response to stress. Particular emphasis will be given to the adaptative process associated with cardiac hypoxia as well as the remodeling events linked to cardiac hypertrophy and heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► A-kinase anchoring proteins (AKAPs) assemble multienzyme signaling complexes in cells. ► AKAPs coordinate signaling cascades that regulate the cardiac response to stress. ► We analyze recent literature describing the role of AKAPs in cardiac pathophysiology. ► We discuss the role of AKAPs in the hypoxic heart and in cardiac remodeling.
Keywords: A kinase anchoring protein (AKAP); Protein kinase A; Cardiac remodeling; Signaling; Cardiomyocyte;

Neuregulin (Nrg)/ErbB and integrin signaling pathways are critical for the normal function of the embryonic and adult heart. Both systems activate several downstream signaling pathways, with different physiological outputs: cell survival, fibrosis, excitation–contraction coupling, myofilament structure, cell–cell and cell–matrix interaction. Activation of ErbB2 by Nrg1β in cardiomycytes or its overexpression in cancer cells induces phosphorylation of FAK (Focal Adhesion Kinase) at specific sites with modulation of survival, invasion and cell–cell contacts. FAK is also a critical mediator of integrin receptors, converting extracellular matrix alterations into intracellular signaling. Systemic FAK deletion is lethal and is associated with left ventricular non-compaction whereas cardiac restriction in adult hearts is well tolerated. Nevertheless, these hearts are more susceptible to stress conditions like trans-aortic constriction, hypertrophy, and ischemic injury. As FAK is both downstream and specifically activated by integrins and Nrg-1β, here we will explore the role of FAK in the heart as a protective factor and as possible mediator of the crosstalk between the ErbB and Integrin receptors. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► Nrg/ErbB signaling is critical for cardiac protection under condition of stress. ► Nrg/ErbB activates FAK signaling pathway in cardiac and cancer cells. ► FAK is critical for cardiac differentiation and survival under condition of stress. ► FAK mediated mechanical signal transduction initiated by integrins. ► These observations suggest a possible ErbB/integrin cross-talk mediated by FAK.
Keywords: Neuregulin; Nrg1β; FAK; Integrin; Cardiomyocyte; Heart;

The cardiac valves are targets of both congenital and acquired diseases. The formation of valves during embryogenesis (i.e., valvulogenesis) originates from endocardial cells lining the myocardium. These cells undergo an endothelial–mesenchymal transition, proliferate and migrate within an extracellular matrix. This leads to the formation of bilateral cardiac cushions in both the atrioventricular canal and the outflow tract. The embryonic origin of both the endocardium and prospective valve cells is still elusive. Endocardial and myocardial lineages are segregated early during embryogenesis and such a cell fate decision can be recapitulated in vitro by embryonic stem cells (ESC). Besides genetically modified mice and ex vivo heart explants, ESCs provide a cellular model to study the early steps of valve development and might constitute a human therapeutic cell source for decellularized tissue-engineered valves. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► The cardiac valves are targets of both congenital and acquired diseases ► Valves form following EMT of endocardial cells and growth of cardiac cushions ► The precardiac mesoderm and the 2nd heart field contribute to the endocardium and cushions. ► Embryonic stem cells (ESC) can be used to better understand human valvulogenesis ► ESCs derivatives could be used in valve repair
Keywords: Valvulogenesis; Atrioventricular canal; Stem cell; EMT;

Small and long non-coding RNAs in cardiac homeostasis and regeneration by Samir Ounzain; Stefania Crippa; Thierry Pedrazzini (923-933).
Cardiovascular diseases and in particular heart failure are major causes of morbidity and mortality in the Western world. Recently, the notion of promoting cardiac regeneration as a means to replace lost cardiomyocytes in the damaged heart has engendered considerable research interest. These studies envisage the utilization of both endogenous and exogenous cellular populations, which undergo highly specialized cell fate transitions to promote cardiomyocyte replenishment. Such transitions are under the control of regenerative gene regulatory networks, which are enacted by the integrated execution of specific transcriptional programs. In this context, it is emerging that the non-coding portion of the genome is dynamically transcribed generating thousands of regulatory small and long non-coding RNAs, which are central orchestrators of these networks. In this review, we discuss more particularly the biological roles of two classes of regulatory non-coding RNAs, i.e. microRNAs and long non-coding RNAs, with a particular emphasis on their known and putative roles in cardiac homeostasis and regeneration. Indeed, manipulating non-coding RNA-mediated regulatory networks could provide keys to unlock the dormant potential of the mammalian heart to regenerate. This should ultimately improve the effectiveness of current regenerative strategies and discover new avenues for repair. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► Cardiac regeneration relies on mobilization of endogenous cardiac progenitors. ► Alternatively, delivery of exogenous stem cells can be envisaged. ► A clear understanding of the cardiac gene regulatory network is needed. ► The cardiac gene regulatory network controls cardiac specification and differentiation. ► Small and large non-coding RNAs control the cardiac gene regulatory network.
Keywords: Non-coding RNA; miRNA; lncRNA; Heart failure; Regeneration; Cardiac progenitor cell;

GM-CSF promotes inflammatory dendritic cell formation but does not contribute to disease progression in experimental autoimmune myocarditis by Przemyslaw Blyszczuk; Silvia Behnke; Thomas F. Lüscher; Urs Eriksson; Gabriela Kania (934-944).
Granulocyte macrophage-colony stimulating factor (GM-CSF) is critically required for the induction of experimental autoimmune myocarditis (EAM), a model of post-inflammatory dilated cardiomyopathy. Its specific role in the progression of myocarditis into end stage heart failure is not known.BALB/c mice were immunized with myosin peptide and complete Freund's adjuvant at days 0 and 7. Heart-infiltrating inflammatory CD133+ progenitors were isolated from inflamed hearts at the peak of inflammation (day 21). In the presence of GM-CSF, inflammatory CD133+ progenitors up-regulated integrin, alpha X (CD11c), class II major histocompatibility complex, CD80 and CD86 co-stimulatory molecules reflecting an inflammatory dendritic cell (DC) phenotype. Inflammatory DCs stimulated antigen-specific CD4+ T cell proliferation and induced myocarditis after myosin peptide loading and adoptive transfer in healthy mice. Moreover, GM-CSF treatment of mice after the peak of disease, between days 21 and 29 of EAM, transiently increased accumulation of inflammatory DCs in the myocardium. Importantly, bone marrow-derived CD11b+ monocytes, rather than inflammatory CD133+ progenitors represent the dominant cellular source of heart-infiltrating inflammatory DCs in EAM. In contrast, GM-CSF treatment neither affected numbers of heart-infiltrating CD45+ and CD3+ T cells nor the development of post-inflammatory fibrosis.GM-CSF treatment promotes formation of inflammatory DCs in EAM. In contrast to the active roles of GM-CSF and DCs in EAM induction, GM-CSF-induced inflammatory DCs neither prevent resolution of active inflammation, nor contribute to post-inflammatory cardiac remodelling. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► GM-CSF is required for the induction of experimental autoimmune myocarditis (EAM). ► The role of GM-CSF in post-inflammatory phase of EAM is not known. ► GM-CSF differentiates inflammatory progenitors into functional dendritic cells. ► GM-CSF promotes accumulation of inflammatory DCs in post-inflammatory myocardium. ► Inflammatory DCs do not contribute to post-inflammatory pathological changes in the heart.
Keywords: GM-CSF; Myocarditis; Inflammatory progenitors; Inflammatory dendritic cells;

Fibroblasts in post-infarction inflammation and cardiac repair by Wei Chen; Nikolaos G. Frangogiannis (945-953).
Fibroblasts are the predominant cell type in the cardiac interstitium. As the main matrix-producing cells in the adult mammalian heart, fibroblasts maintain the integrity of the extracellular matrix network, thus preserving geometry and function. Following myocardial infarction fibroblasts undergo dynamic phenotypic alterations and direct the reparative response. Due to their strategic location, cardiac fibroblasts serve as sentinel cells that sense injury and activate the inflammasome secreting cytokines and chemokines. During the proliferative phase of healing, infarct fibroblasts undergo myofibroblast transdifferentiation forming stress fibers and expressing contractile proteins (such as α-smooth muscle actin). Mechanical stress, transforming growth factor (TGF)-β/Smad3 signaling and alterations in the composition of the extracellular matrix induce acquisition of the myofibroblast phenotype. In the highly cellular and growth factor-rich environment of the infarct, activated myofibroblasts produce matrix proteins, proteases and their inhibitors regulating matrix metabolism. As the infarct matures, “stress-shielding” of myofibroblasts by the cross-linked matrix and growth factor withdrawal may induce quiescence and ultimately cause apoptotic death. Because of their critical role in post-infarction cardiac remodeling, fibroblasts are promising therapeutic targets following myocardial infarction. However, the complexity of fibroblast functions and the pathophysiologic heterogeneity of post-infarction remodeling in the clinical context discourage oversimplified approaches in clinical translation. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.► In normal hearts cardiac fibroblasts maintain the integrity of the extracellular matrix. ► In the infarcted heart fibroblasts activate the inflammasome and secrete cytokines. ► In the healing infarct fibroblasts transdifferentiate into myofibroblasts. ► Myofibroblasts secrete extracellular matrix proteins and regulate matrix metabolism. ► As the infarct matures, myofibroblasts become quiescent and undergo apoptosis.
Keywords: Fibroblast; Inflammation; Growth factors; TGF-β; Cardiac remodeling; Extracellular matrix;