Cardiomyocyte dysfunction in inherited cardiomyopathies

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

Genetic restrictive cardiomyopathy: causes and consequences

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

Interventricular differences of signaling pathways-mediated regulation of cardiomyocyte function in response to high oxidative stress in the post-ischemic failing rat heart

Standard heart failure (HF) therapies have failed to improve cardiac function or survival in HF patients with right ventricular (RV) dysfunction suggesting a divergence in the molecular mechanisms of RV vs. left ventricular (LV) failure. Here we aimed to investigate interventricular differences in sarcomeric regulation and function in experimental myocardial infarction (MI)-induced HF with reduced LV ejection fraction (HFrEF). MI was induced by LAD ligation in Sprague–Dawley male rats. Sham-operated animals served as controls. Eight weeks after intervention, post-ischemic HFrEF and Sham animals were euthanized. Heart tissue samples were deep-frozen stored ($\it n$ = 3–5 heart/group) for ELISA, kinase activity assays, passive stiffness and $Ca^{2+}$-sensitivity measurements on isolated cardiomyocytes, phospho-specific Western blot, and PAGE of contractile proteins, as well as for collagen gene expressions. Markers of oxidative stress and inflammation showed interventricular differences in post-ischemic rats: TGF-$\beta$1, lipid peroxidation, and 3-nitrotyrosine levels were higher in the LV than RV, while hydrogen peroxide, VCAM-1, TNF$\alpha$, and TGF-$\beta$1 were increased in both ventricles. In addition, nitric oxide (NO) level was significantly decreased, while FN-1 level was significantly increased only in the LV, but both were unchanged in RV. CaMKII activity showed an 81.6% increase in the LV, in contrast to a 38.6% decrease in the RV of HFrEF rats. Cardiomyocyte passive stiffness was higher in the HFrEF compared to the Sham group as evident from significantly steeper $F_{passive}$ vs. sarcomere length relationships. In vitro treatment with CaMKII$\delta$, however, restored cardiomyocyte passive stiffness only in the HFrEF RV, but had no effect in the HFrEF LV. PKG activity was lower in both ventricles in the HFrEF compared to the Sham group. In vitro PKG administration decreased HFrEF cardiomyocyte passive stiffness; however, the effect was more pronounced in the HFrEF LV than HFrEF RV. In line with this, we observed distinct changes of titin site-specific phosphorylation in the RV vs. LV of post-ischemic rats, which may explain divergent cardiomyocyte stiffness modulation observed. Finally, $Ca^{2+}$-sensitivity of RV cardiomyocytes was unchanged, while LV cardiomyocytes showed increased $Ca^{2+}$-sensitivity in the HFrEF group. This could be explained by decreased Ser-282 phosphorylation of cMyBP-C by 44.5% in the RV, but without any alteration in the LV, while Ser-23/24 phosphorylation of cTnI was decreased in both ventricles in the HFrEF vs. the Sham group. Our data pointed to distinct signaling pathways-mediated phosphorylations of sarcomeric proteins for the RV and LV of the post-ischemic failing rat heart. These results implicate divergent responses for oxidative stress and open a new avenue in targeting the RV independently of the LV

Soluble adenylyl cyclase

$\bf Aims$ In contrast to the membrane bound adenylyl cyclases, the soluble adenylyl cyclase (sAC) is activated by bicarbonate and divalent ions including calcium. sAC is located in the cytosol, nuclei and mitochondria of several tissues including cardiac muscle. However, its role in cardiac pathology is poorly understood. Here we investigate whether sAC is involved in hypertrophic growth using two different model systems. $\textbf {Methods and results}$ In isolated adult rat cardiomyocytes hypertrophy was induced by 24 h $\beta_{1}$-adrenoceptor stimulation using isoprenaline (ISO) and a $\beta_{2}$-adrenoceptor antagonist (ICI118,551). To monitor hypertrophy cell size along with RNA/DNA- and protein/DNA ratios as well as the expression level of α-skeletal actin were analyzed. sAC activity was suppressed either by treatment with its specific inhibitor KH7 or by knockdown. Both pharmacological inhibition and knockdown blunted hypertrophic growth and reduced expression levels of α-skeletal actin in ISO/ICI treated rat cardiomyocytes. To analyze the underlying cellular mechanism expression levels of phosphorylated CREB, B-Raf and Erk1/2 were examined by western blot. The results suggest the involvement of B-Raf, but not of Erk or CREB in the pro-hypertrophic action of sAC. In wild type and sAC knockout mice pressure overload was induced by transverse aortic constriction. Hemodynamics, heart weight and the expression level of the atrial natriuretic peptide were analyzed. In accordance, transverse aortic constriction failed to induce hypertrophy in sAC knockout mice. Mechanistic analysis revealed a potential role of Erk1/2 in TAC-induced hypertrophy. $\bf Conclusion$ Soluble adenylyl cyclase might be a new pivotal player in the cardiac hypertrophic response either to long-term $\beta_{1}$-adrenoceptor stimulation or to pressure overload

De novo missense mutations in TNNC1\it TNNC1 and TNNI3\it TNNI3 causing severe infantile cardiomyopathy affect myofilament structure and function and are modulated by troponin targeting agents

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 $\it TNNC1$ ($\it {p.cTnC-G34S}$) and $\it TNNI3$ (($\it {p.cTnI-D127Y}$) 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 inter-actions, 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

The interplay between S-glutathionylation and phosphorylation of cardiac troponin I and myosin binding protein C in end-stage human failing hearts

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 $Ca^{2+}$-activated tension and $Ca^{2+}$ 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 $Ca^{2+}$ 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

Infantile restrictive cardiomyopathy

$\it TNNI3$ encoding cTnI, the inhibitory subunit of the troponin complex, is the main target for mutations leading to restrictive cardiomyopathy (RCM). Here we investigate two cTnI-R170G/W amino acid replacements, identified in infantile RCM patients, which are located in the regulatory C-terminus of cTnI. The C-terminus is thought to modulate the function of the inhibitory region of cTnI. Both cTnI-R170G/W strongly enhanced the $Ca^{2+}$-sensitivity of skinned fibres, as is typical for RCM-mutations. Both mutants strongly enhanced the affinity of troponin (cTn) to tropomyosin compared to wildtype cTn, whereas binding to actin was either strengthened (R170G) or weakened (R170W). Furthermore, the stability of reconstituted thin filaments was reduced as revealed by electron microscopy. Filaments containing R170G/W appeared wavy and showed breaks. Decoration of filaments with myosin subfragment S1 was normal in the presence of R170W, but was irregular with R170G. Surprisingly, both mutants did not affect the $Ca^{2+}$-dependent activation of reconstituted cardiac thin filaments. In the presence of the N-terminal fragment of cardiac myosin binding protein C (cMyBPC-C0C2) cooperativity of thin filament activation was increased only when the filaments contained wildtype cTn. No effect was observed in the presence of cTn containing R170G/W. cMyBPC-C0C2 significantly reduced binding of wildtype troponin to actin/tropomyosin, but not of both mutant cTn. Moreover, we found a direct troponin/cMyBPC-C0C2 interaction using microscale thermophoresis and identified cTnI and cTnT, but not cTnC as binding partners for cMyBPC-C0C2. Only cTn containing cTnI-R170G showed a reduced affinity towards cMyBPC-C0C2. Our results suggest that the RCM cTnI variants R170G/W impair the communication between thin and thick filament proteins and destabilize thin filaments