The sarcomere as the smallest contractile unit is prone to alterations in its functional, structural and associated proteins. Sarcomeric dysfunction leads to heart failure or cardiomyopathies like hypertrophic (HCM) or restrictive cardiomyopathy (RCM) etc. Genetic based RCM, a very rare but severe disease with a high mortality rate, might be induced by mutations in genes of non-sarcomeric, sarcomeric and sarcomere associated proteins. In this review, we discuss the functional effects in correlation to the phenotype and present an integrated model for the development of genetic RCM.

Oxidative stress is defined as an imbalance between the antioxidant defense system and the production of reactive oxygen species (ROS). At low levels, ROS are involved in the regulation of redox signaling for cell protection. However, upon chronical increase in oxidative stress, cell damage occurs, due to protein, DNA and lipid oxidation. Here, we investigated the oxidative modifications of myofilament proteins, and their role in modulating cardiomyocyte function in end-stage human failing hearts. We found altered maximum Ca²⁺-activated tension and Ca²⁺ sensitivity of force production of skinned single cardiomyocytes in end-stage human failing hearts compared to non-failing hearts, which was corrected upon treatment with reduced glutathione enzyme. This was accompanied by the increased oxidation of troponin I and myosin binding protein C, and decreased levels of protein kinases A (PKA)- and C (PKC)-mediated phosphorylation of both proteins. The Ca²⁺ sensitivity and maximal tension correlated strongly with the myofilament oxidation levels, hypo-phosphorylation, and oxidative stress parameters that were measured in all the samples. Furthermore, we detected elevated titin-based myocardial stiffness in HF myocytes, which was reversed by PKA and reduced glutathione enzyme treatment. Finally, many oxidative stress and inflammation parameters were significantly elevated in failing hearts compared to non-failing hearts, and corrected upon treatment with the anti-oxidant GSH enzyme. Here, we provide evidence that the altered mechanical properties of failing human cardiomyocytes are partially due to phosphorylation, S-glutathionylation, and the interplay between the two post-translational modifications, which contribute to the development of heart failure.

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in TNNC1 (p.cTnC-G34S) and TNNI3 (p.cTnI-D127Y) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit interactions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient’s myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations.

We analyzed actin cytoskeleton alterations during NET extrusion by neutrophil-like dHL-60 cells and human neutrophils in the absence of DNase1 containing serum to avoid chromatin degradation and microfilament disassembly. NET-formation by dHL-60 cells and neutrophils was induced by Ionomycin or phorbol-12-myristat-13-acetate (PMA). Subsequent staining with anti-actin and TRITC-phalloidin showed depolymerization of the cortical F-actin at spatially confined areas, the NET extrusion sites, effected by transient activation of the monooxygenase MICAL-1 supported by the G-actin binding proteins cofilin, profilin, thymosin ß4 and probably the F-actin fragmenting activity of gelsolin and/or its fragments, which also decorated the formed NETs. MICAL-1 itself appeared to be proteolyzed by neutrophil elastase possibly to confine its activity to the NET-extrusion area. The F-actin oxidization activity of MICAL-1 is inhibited by Levosimendan leading to reduced NET-formation. Anti-gasdermin-D immunohistochemistry showed a cytoplasmic distribution in non-stimulated cells. After stimulation the NET-extrusion pore displayed reduced anti-gasdermin-D staining but accumulated underneath the plasma membrane of the remaining cell body. A similar distribution was observed for myosin that concentrated together with cortical F-actin along the periphery of the remaining cell body suggesting force production by acto-myosin interactions supporting NET expulsion as indicated by the inhibitory action of the myosin ATPase inhibitor blebbistatin. Isolated human neutrophils displayed differences in their content of certain cytoskeletal proteins. After stimulation neutrophils with high gelsolin content preferentially formed "cloud"-like NETs, whereas those with low or no gelsolin formed long "filamentous" NETs.

Inherited cardiomyopathies form a heterogenous group of disorders that affect the structure and function of the heart. Defects in the genes encoding sarcomeric proteins are associated with various perturbations that induce contractile dysfunction and promote disease development. In this review we aimed to outline the functional consequences of the major inherited cardiomyopathies in terms of myocardial contraction and kinetics, and to highlight the structural and functional alterations in some sarcomeric variants that have been demonstrated to be involved in the pathogenesis of the inherited cardiomyopathies. A particular focus was made on mutation-induced alterations in cardiomyocyte mechanics. Since no disease-specific treatments for familial cardiomyopathies exist, several novel agents have been developed to modulate sarcomere contractility. Understanding the molecular basis of the disease opens new avenues for the development of new therapies. Furthermore, the earlier the awareness of the genetic defect, the better the clinical prognostication would be for patients and the better the prevention of development of the disease.

The human mutant cardiac α-actins p.A295S or p.R312H and p.E361G, correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by the baculovirus/Sf21 insect cell system and purified to homogeneity. The purified cardiac actins maintained their native state but showed differences in Ca²⁺-sensitivity to stimulate the myosin-subfragment1 ATPase. Here we analyzed the interactions of these c-actins with actin-binding and -modifying proteins implicated in cardiomyocyte differentiation. We demonstrate that Arp2/3 complex and the formin mDia3 stimulated the polymerization rate and extent of the c-actins, albeit to different degrees. In addition, we tested the effect of the MICAL-1 monooxygenase, which modifies the supramolecular actin organization during development and adaptive processes. MICAL-1 oxidized these c-actin variants and induced their de-polymerization, albeit at different rates. Transfection experiments using MDCK cells demonstrated the preferable incorporation of wild type and p.A295S c-actins into their microfilament system but of p.R312H and p.E361G actins into the submembranous actin network. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding HA-tagged c-actin variants showed their incorporation into microfilaments after one day in culture and thereafter into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferentially incorporated at the minus ends.