and thereby elevates intracellular Na þ , which in turn elevates Ca 2 þ by hampering cytosolic Ca 2 þ export via the forward mode Na þ /Ca 2 þ exchanger (NCX) and may increase reverse mode NCX-mediated Ca 2 þ influx during the early phase of the action potential. Istaroxime has similar effects as digitalis but also activates SERCA to accelerate diastolic Ca 2 þ uptake into the SR. SERCA2a gene therapy increases the mRNA and protein levels of SERCA and thereby, potentiates SR Ca 2 þ uptake and release. Nitroxyl (HNO) activates SERCA activity and increases myofilament Ca 2 þ sensitivity. Levosimendan increases the affinity of troponin C to Ca 2 þ and thereby, increases force for any cytosolic Ca 2 þ concentration. levosimendan inhibits PDE3, which elevates cAMP and PKA activity with subsequent effects on Ca 2 þ handling for catecholamines and PDE-inhibitors. Omecamtiv mecarbil is a myosin activator that prolongs actin–myosin interaction and thereby, results in a prolongation (but not acceleration) of contraction. Elamipretide (also known as Bendavia or MTP-131) binds to cardiolipin in the inner mitochondrial membrane, increasing ATP production and reducing the generation of reactive oxygen species (ROS). Trimetazidine and perhexiline optimize substrate utilization and thereby, improve cardiac energetics. Iron restores iron deficiency and thereby, may improve function of Krebs cycle enzymes and possibly, the electron transport (ETC).

phenotype in mdx mice. These findings suggest that SLN might be a novel target for DMD therapy. Obscurin was discovered as binding partner of titin and Novex-3, a titin splice variant. Although their direct binding is known for [ 15 years, the physiological relevance of their interaction has been elu- sive. To assess the effects of the obscurin/titin binding in vivo, we generated a deletion model, Obscn- D Ig58/59, that carries truncated obscurin lacking the Ig58/Ig59 region that supports binding to both titin and Novex-3. Homozygous Obscn- D Ig58/59 male mice develop left ventricular (LV) hypertrophy by 6 months, which progresses to LV dilation and severe arrhythmia by 1 year, while female mice present mild arrhythmia. Exertion of pathological and physiological stress in young mice via b -adrenergic stimulation and strenuous exercise, respectively, revealed electrical abnormalities and poorer running ability. Mutations in obscurin and titins, including ones that disrupt their binding, are linked to cardiac and skeletal myopathies. It is thus apparent that the obscurin/titin complex is essential for normal muscle structure and function, and that disruption of their binding is associated with muscle pathogenicity. Our findings using the Obscn- D Ig58/59 model corroborate this notion. Aims : Increased Ankrd1 levels linked to genetic mutations have been correlated to congenital heart disease onset and adult cardiomyopathy occurrence in humans. The link between increased ANKRD1 level and cardiac structural and functional disease onset is not understood. To get insight into this problem, we have generated a ANKRD1 mouse model by overexpressing ANKRD1 in the myocardium. Methods and Results : We show that ANKRD1 delineates discrete sub-compartments in the developing mouse heart. ANKRD1 trans- genic mice present impaired cardiac remodeling, which strongly affects the developing sinoatrial region and leads to sinus venosus defects. Transgenic mice survive to adulthood but develop left atrial enlargement accompanied by severe diastolic dysfunction. Embryonic and neonatal transgenic cardiomyocytes present irregular shape and sarcomeric disorganization, which progresses into sarcomeric loss and mitochondrial damage in adult ventricular but not atrial cardiomy- ocytes. While isolated embryonic transgenic myofibrils show the same mechanical properties of wild type samples, neonatal transgenic myofibrils present higher passive tension and maximal force com-pared to wild type. This indicates the presence in ANKRD1 transgenic mice of a faster functional shift towards stiffer and hyper-contractile cardiomyocytes, triggered by the increase in workload at birth. At the molecular level, these changes are accompanied by dynamic alterations in titin isoforms ratio. Interestingly, adult wild type and transgenic myofibrils show the same passive tension as transgenic neonatal myofibrils, with adult transgenic myofibrils showing a higher maximal force accompanied at this stage by a marked slowing down of the relaxation phase compatible with the overt diastolic disfunction of adult ANKRD1 transgenic mice. Conclusions : Our data indicate that genetic mutations leading to increased ANKRD1 levels can lead both to congenital heart disease and adult cardiomyopathy via a common cellular mechanism, with ANKRD1 playing the role of a critical strain sensor-signaling mole-cule finely modulating cardiomyocyte function during development and postnatal life. The giant protein titin spans from the Z-disk to the M-line in the sarcomere of striated muscle cells, where it functions as a molecular spring during stretching and relaxation. In this study we used immuno electron microscopy and three-dimensional reconstruction to localize full-length titin and Novex-3 (‘‘tiny titin’’) at different stages of myofibrillogenesis in regenerating rat soleus muscle after notexin-induced myofibril breakdown. Two days after intoxication with notexin we observed first single thick filaments in the cytosol colo-calized with full length titin. In addition, we identified subcellular compartments containing Novex-3 titin as integral elements of emerging Z-bodies. Thick filaments aligned to build first premyofib- rils containing titin, myosin and Z-bodies; three days after intoxication we found Z-bodies fusing to Z-disks, forming contracted sarcomeric structures, which later develop into mature myofibrils with I- and A-Band showing the typical striation pattern. Our results support a model in which titin acts as a molecular scaffold for the assembly of Z-discs and thick filaments during skeletal muscle regeneration. area of intensive study despite over a decade of frustratingly slow progress and modest clinical efficacy. A fundamental limitation in myocardial regeneration is inherently poor reparative capacity of adult mammalian heart which declines over lifespan. Augmentation of repair requires unnatural solutions to overcome normal adult myocardial biology using Regeneration Associated Cellular Effectors (RACE) to deliver func- tionally competent therapeutic interventions. The logic and rationale

Mutations in the FLNC gene cause familial and sporadic myopathies and cardiomyopathies with marked clinical variability. Filamin-C is mainly expressed in striated muscles and localizes to Z-discs, myotendinous junctions, and intercalated discs. The most frequent human mutation, p.W2710X, which is associated with a myofibrillar myopathy phenotype, deletes the carboxy-terminal 16 amino acids from filamin-C, and abolishes its dimerization property. Here, we analyzed mice homozygous for this mutation, i.e. p.W2711X in mice. These mice are viable and fertile. Although they express highly reduced levels of solely mutant filamin-C protein they show only mild myopathic features. Mutant filamin-C displayed marked re-localization from Z-discs to mechanically strained parts of the muscle cells, i.e. myotendinous junctions and filamin-C and Xin-positive myofibrillar lesions streaming between Z-discs. Ultrastructural studies confirmed that lesions are filamentous and structured, and not amorphous aggregates. Our data demonstrate that filamin-C dimerization is not required for assembly and basic function of myofibrils, but rather for mechanosensing in the context of Z-disc stabilization and maintenance.

AimsWe investigated newly generated immortalized heterozygous and homozygous R349P desmin knock‐in myoblasts in conjunction with the corresponding desminopathy mice as models for desminopathies to analyse major protein quality control processes in response to the presence of R349P mutant desmin.MethodsWe used hetero‐ and homozygous R349P desmin knock‐in mice for analyses and for crossbreeding with p53 knock‐out mice to generate immortalized R349P desmin knock‐in skeletal muscle myoblasts and myotubes. Skeletal muscle sections and cultured muscle cells were investigated by indirect immunofluorescence microscopy, proteasomal activity measurements and immunoblotting addressing autophagy rate, chaperone‐assisted selective autophagy and heat shock protein levels. Muscle sections were further analysed by transmission and immunogold electron microscopy.ResultsWe demonstrate that mutant desmin (i) increases proteasomal activity, (ii) stimulates macroautophagy, (iii) dysregulates the chaperone assisted selective autophagy and (iv) elevates the protein levels of αB‐crystallin and Hsp27. Both αB‐crystallin and Hsp27 as well as Hsp90 displayed translocation patterns from Z‐discs as well as Z‐I junctions, respectively, to the level of sarcomeric I‐bands in dominant and recessive desminopathies.ConclusionsOur findings demonstrate that the presence of R349P mutant desmin causes a general imbalance in skeletal muscle protein homeostasis via aberrant activity of all major protein quality control systems. The augmented activity of these systems and the subcellular shift of essential heat shock proteins may deleteriously contribute to the previously observed increased turnover of desmin itself and desmin‐binding partners, which triggers progressive dysfunction of the extrasarcomeric cytoskeleton and the myofibrillar apparatus in the course of the development of desminopathies.

Michael Schwarzl, Nazha Hamdani, Sebastian Seiler, Alessio Alogna, Martin Manninger, Svetlana Reilly, Birgit Zirngast, Alexander Kirsch, Paul Steendijk, Jochen Verderber, David Zweiker, Philipp Eller, Gerald Höfler, Silvia Schauer, Kathrin Eller, Heinrich Maechler, Burkert M. Pieske, Wolfgang A. Linke, Barbara Casadei, and Heiner Post Department of General and Interventional Cardiology, University Heart Center Hamburg-Eppendorf, Hamburg, Germany; Department of Cardiovascular Physiology, Ruhr University Bochum, Bochum, Germany; Division of General Medicine, Klinikum Starnberg, Starnberg, Germany; Division of Cardiology, Department of Internal Medicine, Medical University of Graz, Graz, Austria; Division of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; Department of Cardiothoracic Surgery, Medical University of Graz, Graz, Austria; Division of Nephrology, Department of Internal Medicine, Medical University of Graz, Graz, Austria; Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands; Intensive Care Unit, Department of Internal Medicine, Medical University of Graz, Graz, Austria; Department of Pathology, Medical University of Graz, Graz, Austria; Division of Cardiology, Medical Department, Charité Berlin Campus Virchow, Berlin, Germany

Neuregulin (NRG)-1 is involved in the preservation of left ventricular performance. Nevertheless, the role of NRG-1 in pulmonary arterial hypertension(PAH) and right ventricular(RV) diastolic stiffness is unknown. We analysed the presence and possible underlying mechanisms of RV diastolic dysfunction in an animal model of PAH and the role of NRG-1 in this context.

In myocytes, small heat shock proteins (sHSPs) are preferentially translocated under stress to the sarcomeres. The functional implications of this translocation are poorly understood. We show here that HSP27 and αB-crystallin associated with immunoglobulin-like (Ig) domain-containing regions, but not the disordered PEVK domain (titin region rich in proline, glutamate, valine, and lysine), of the titin springs. In sarcomeres, sHSP binding to titin was actin filament independent and promoted by factors that increased titin Ig unfolding, including sarcomere stretch and the expression of stiff titin isoforms. Titin spring elements behaved predominantly as monomers in vitro. However, unfolded Ig segments aggregated, preferentially under acidic conditions, and αB-crystallin prevented this aggregation. Disordered regions did not aggregate. Promoting titin Ig unfolding in cardiomyocytes caused elevated stiffness under acidic stress, but HSP27 or αB-crystallin suppressed this stiffening. In diseased human muscle and heart, both sHSPs associated with the titin springs, in contrast to the cytosolic/Z-disk localization seen in healthy muscle/heart. We conclude that aggregation of unfolded titin Ig domains stiffens myocytes and that sHSPs translocate to these domains to prevent this aggregation.

Eine sicher nachgewiesene Funktion von Titin ist sein Beitrag zur passiven Spannung (Fpassiv), wofür seine elastische Region verantwortlich ist. Es wird vermutet, dass Titin insbesondere bei Herzinsuffizienz mit erhaltener Ejektionsfraktion (heart failure with preserved ejection fraction, HFpEF) zur diastolischen Dysfunktion des linken Ventrikels (LV) beiträgt. Die Titin-basierte Steifigkeit kann durch Wechsel der Isoformen oder durch Phosphorylierung moduliert werden.