De Novo Missense Mutations in TNNC1 and 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 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.

Genetic Restrictive Cardiomyopathy: Causes and Consequences-An Integrative Approach.

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.

Infantile restrictive cardiomyopathy: cTnI-R170G/W impair the interplay of sarcomeric proteins and the integrity of thin filaments.

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 Ca2+-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 Ca2+-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.

Soluble adenylyl cyclase: A novel player in cardiac hypertrophy induced by isoprenaline or pressure overload.

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. METHODS AND RESULTS: In isolated adult rat cardiomyocytes hypertrophy was induced by 24 h ß1-adrenoceptor stimulation using isoprenaline (ISO) and a ß2-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. CONCLUSION: Soluble adenylyl cyclase might be a new pivotal player in the cardiac hypertrophic response either to long-term ß1-adrenoceptor stimulation or to pressure overload.