ARVC-related mutations in divergent region 3 alter functional properties of the cardiac ryanodine receptor.

Two single-nucleotide polymorphisms in the type 2 ryanodine receptor (RyR2) leading to the nonsynonymous amino acid replacements G1885E and G1886S are associated with arrhythmogenic right ventricular cardiomyopathy in patients who are carrying both of the corresponding RyR2 alleles. The functional properties of HEK293 cell lines isogenically expressing RyR2 mutants associated with arrhythmogenic right ventricular cardiomyopathy, RyR2-G1885E, RyR2-G1886S, RyR2-G1886D (mimicking a constitutively phosphorylated Ser(1886)), and the double mutant RyR2-G1885E/G1886S were investigated by analyzing the intracellular Ca(2+) release activity resulting from store-overload-induced calcium release. The substitution of serine for Gly(1886) caused a significant increase in the cellular Ca(2+) oscillation activity compared with RyR2 wild-type-expressing HEK293 cells. It was even more pronounced if glycine 1885 or 1886 was replaced by the acidic amino acids glutamate (G1885E) or aspartate (G1886D). Surprisingly, when both substitutions were introduced in the same RyR2 subunit (RyR2-G1885E/G1886S), the store-overload-induced calcium release activity was nearly completely abolished, although the Ca(2+) loading of the intracellular stores was markedly enhanced, and the channel still displayed substantial Ca(2+) release on stimulation by 5 mM caffeine. These results suggest that the adjacent glycines 1885 and 1886, located in the divergent region 3, are critical for the function and regulation of RyR2.

Composite polymorphisms in the ryanodine receptor 2 gene associated with arrhythmogenic right ventricular cardiomyopathy.

OBJECTIVE: Mutations in the cardiac ryanodine receptor (RYR2) gene have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC). The molecular mechanisms by which genetic modifications lead to ARVC are still not well understood. METHODS: ARVC patients were screened for mutations in the RYR2 gene by denaturing HPLC and DNA sequencing. Single channel measurements were carried out with RyR2 channels purified from explanted hearts of ARVC patients. RESULTS: None of the published RYR2 mutations were found in our ARVC-cohort. However, we identified two single nucleotide polymorphisms (SNPs) in exon 37 of the human RYR2 gene which lead to the amino acid exchanges G1885E and G1886S, respectively. Both SNPs together were found exclusively in 3 out of 85 ARVC patients in a composite heterozygous fashion (genotype T4). This genotype was associated with ARVC (p<0.05) but not with dilated cardiomyopathy (DCM, 79 patients) or none-failing controls (463 blood donors). However, either one of the two SNPs were identified in further 7 ARVC patients, in 11 DCM patients, and in 64 blood donors. The SNP leading to G1886S may create a protein kinase C phosphorylation site in the human RyR2. Single channel recordings at pCa4.3 revealed four conductance states for the RyR2 of genotype T4 and a single open state for the wild type RyR2. At pCa7.7, the lowest subconductance state of the RyR2 channel of genotype T4 persisted with a greatly enhanced open probability indicating a leaky channel. CONCLUSION: The RyR2 channel leak under diastolic conditions could cause SR-Ca2+ depletion, concomitantly arrhythmogenesis and heart failure in a subgroup of ARVC patients of genotype T4. A change in the RyR2 subunit composition due to the combined expression of both SNPs alters the behaviour of the tetrameric channel complex.

Phosphorylation of human cardiac troponin I G203S and K206Q linked to familial hypertrophic cardiomyopathy affects actomyosin interaction in different ways.

cAMP-dependent protein kinase (PKA)-dependent phosphorylation of the two serine residues in the amino terminal region unique to cardiac troponin I (cTnI) is known to cause two effects: (i) decrease of the maximum Ca2+-controlled thin filament-activated myosin S1-ATPase (actoS1-ATPase) activity and mean sliding velocity of reconstituted thin filaments; (ii) rightward shift of the Ca2+ activation curves of actoS1-ATPase activity, filament sliding velocity, and force generation. We have studied the influence of phosphorylation of human wild-type cTnI and of two mutant cTnI (G203S and K206Q) causing familial hypertrophic cardiomyopathy (fHCM) on the secondary structure by circular dichroism spectroscopy and on the Ca2+ regulation of actin-myosin interaction using actoS1-ATPase activity and in vitro motility assays. Both mutations slightly influence the backbone structure of cTnI but only the secondary structure of cTnI-G203S is also affected by bis-phosphorylation of cTnI. In functional studies, cTnI-G203S behaves similarly to wild-type cTnI, i.e. the mutation itself has no measurable effect and bis-phosphorylation alters the actoS1-ATPase activity and the in vitro thin filament motility in the same way as does bis-phosphorylation of wild-type cTnI. In contrast, the mutation K206Q leads to a considerable increase in the maximum actoS1-ATPase activity as well as filament motility compared to wild-type cTnI. Bis-phosphorylation of this mutant cTnI still suppresses the maximum actoS1-ATPase activity and filament sliding velocity but does no longer affect the Ca2+ sensitivity of these processes. Thus, these two fHCM-linked cTnI mutations, although reflecting similar pathological situations, exert different effects on the actomyosin system per se and in response to bis-phosphorylation of cTnI.

Differential regulation of Ca2+-dependent ATPase-activity in left ventricular myocardium during mechanical circulatory support.

BACKGROUND: Myocardial recovery is observed in some end-stage heart failure patients after mechanical circulatory support. The sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase (Ca2+-ATPase) activity is down-regulated in failing myocardium and contributes to heart failure-associated contraction/relaxation abnormalities. Regulation of Ca(2+)-ATPase after mechanical support was shown to be heterogeneous. Thus, we analyzed Ca(2+)-ATPase activity and protein expression in the paired myocardial samples of 21 patients supported by ventricular assist devices to identify factors that influence restoration of the Ca(2+)-transient after ventricular assist device support. METHODS: We measured Ca(2+)-ATPase activity using a reduced nicotinamide-adenine dinucleotide-coupled reaction, determined sarcoplasmic reticulum Ca(2+)-dependent ATPase protein using Western blotting, and determined 4-hydroxyproline using amino-acid analysis. RESULTS: The mean Ca(2+)-ATPase activity decreased at assist-device implantation and slightly increased at transplantation, but remained significantly lower than in non-failing donor hearts. However, individual responses were heterogeneous. Patients with older age, increased left ventricular diameter, and increased 4-hydroxyproline content showed down-regulation of Ca(2+)-ATPase activity, whereas we found up-regulation in patients with low values for these parameters after assist-device support. CONCLUSIONS: Sarcoplasmic reticulum Ca(2+)-ATPase activity, which influences the myocardial Ca(2+)-transient, generally is not restored to normal values in assist-device-supported hearts, but depends on a combined score of the left ventricular end-diastolic diameter, degree of ventricular fibrosis, and age of the patient at the time of assist-device implantation.