Alternative Splicing of NOX4 in the Failing Human Heart

Increased oxidative stress is a major contributor to the development and progression of heart failure, however, our knowledge on the role of the distinct NADPH oxidase (NOX) isoenzymes, especially on NOX4 is controversial. Therefore, we aimed to characterize NOX4 expression in human samples from healthy and failing hearts. Explanted human heart samples (left and right ventricular, and septal regions) were obtained from patients suffering from heart failure of ischemic or dilated origin. Control samples were obtained from donor hearts that were not used for transplantation. Deep RNA sequencing of the cardiac transcriptome indicated extensive alternative splicing of the NOX4 gene in heart failure as compared to samples from healthy donor hearts. Long distance PCR analysis with a universal 5'-3' end primer pair, allowing amplification of different splice variants, confirmed the presence of the splice variants. To assess translation of the alternatively spliced transcripts we determined protein expression of NOX4 by using a specific antibody recognizing a conserved region in all variants. Western blot analysis showed up-regulation of the full-length NOX4 in ischemic cardiomyopathy samples and confirmed presence of shorter isoforms both in control and failing samples with disease-associated expression pattern. We describe here for the first time that NOX4 undergoes extensive alternative splicing in human hearts which gives rise to the expression of different enzyme isoforms. The full length NOX4 is significantly upregulated in ischemic cardiomyopathy suggesting a role for NOX4 in ROS production during heart failure

Muscle phenotype of the myostatin mutant Compact mice and myostatin/IGF-I transcript levels in pathological human hearts

Myostatin (Mstn) is an important negative regulator of skeletal muscle growth. However, it plays also a crucial role in governing cardiomyocyte growth, heart metabolism and contraction. Our aim was to describe different aspects of Mstn signaling in skeletal muscle and heart tissue. To this end, two different model systems have been used in our experiments: (1) the Mstn mutant Compact (Cmpt)mice and (2) healthy and pathological human hearts. The hypermuscular Cmpt mice carry a 12 - bp natural mutation in the Mstn propeptide, with additional modifier genes being responsible for the phenotype. Muscle cellularity of the fast tibialis anterior (TA) and extensor digitorum longus (EDL) as well as the mixed - type soleus (SOL) muscles of Cmpt and BALB/c mice was examined by immunohistochemical staining of the myosin heavy chain (MHC) proteins. In addition, transcript levels of MHC isoforms were quantified by qRT - PCR. On the other hand, gene expression of Mstn and IGF-I, the two major but mostly counteracting regulators of heart tissue have been investigated in different regions (septum,left and right ventricle s) of healthy or dilated (DCM) and ischemic cardiomyopathic (ICM) patient hearts. A comprehensive qRT -PCR analysis was carried out by measuring the expression of Mstn, its receptor Activin receptor IIB (ActRIIB), IGF-I, IGF-I receptor (IGF-I receptor), as well as microRNA-208, the negative post-transcriptional regulator of Mstn. According to our results all investigated muscles of Cmpt mice were significantly larger compared to wild type characterized by fiber hyperplasia of different grade. Fiber hypertrophy was not present in TA, however, EDL muscles showed specific IIB fiber hypertrophy while the (I and IIA) fibers of SOL muscles wer e generally hypertrophied. Both the fast TA and EDL muscles of Cmpt mice contained significantly more glycolytic IIB fibers accompanied by decreased number of IIX and IIA fibers, however, this was not the case for the SOL muscles. In summary, Cmpt mouse, inspite of its complex genetic background, shows similarities (at least in fast muscles) to Mstn knockout mice in terms of muscle cellularity and glycolytic muscle phenotype, suggesting that the lack of Mstn is responsible for these morphological changes. However, based on the more pronounced hyperplasia in Cmpt fast muscles as well as the different cellularity and oxidative phenotype of Cmpt SOL, additional studies are needed to elucidate the molecular mechanisms of Mstn inactivity and the possible role of modifier genes in Cmpt mice. In our human heart study , we have found that in healthy control hearts the ratio of Mstn/IGF - I signaling was significantly higher in the left ventricle/septum than in the right ventricle. Moreover, Mstn transcript levels were significantly upregulated in all heart regions of DCM but not ICM patients. However, the ratio of Mstn/IGF - I signaling remained increased in the left ventricle/septum compared to the right ventricle of DCM patients (similar to healthy hearts). In contrast, in ICM hearts significant transcript changes were detected mainly in IGF- I signaling. In paralell with these results microRNA -208 showed mild upregulation in the left ventricle of both DCM and ICM hearts.This is the first demonstration of a spatial asymmetry in the expression pattern of Mstn/IGF - I in healthy hearts, which is likely to play a role in the different growth regulation of left vs. right ventricle. Moreover, we identified Mstn as a massively up-regulated gene in DCM but not in ICM as part of potential compensatory mechanisms in the failing heart

Myostatin propeptide mutation of the hypermuscular Compact mice decreases the formation of myostatin and improves insulin sensitivity

The TGF-beta family member myostatin (growth/differentiation factor-8, GDF-8) is a negative regulator of skeletal muscle growth. The hypermuscular Compact mice carry the 12-bp Mstn(Cmpt-dl1Abc) deletion in the sequence encoding the propeptide region of the precursor promyostatin and additional modifier genes of the Compact genetic background contribute to determine the full expression of the phenotype. In this study, by using mice strains carrying mutant or wild-type myostatin alleles with Compact genetic background, and non-mutant myostatin with wild-type background we studied separately the effect of the Mstn(Cmpt-dl1Abc) mutation or the Compact genetic background on morphology, metabolism and signaling. We show that both the Compact myostatin mutation and Compact genetic background account for determination of skeletal muscle size. Despite the increased musculature of Compacts, the absolute size of heart and kidney are not influenced by myostatin mutation; however, the Compact genetic background increases them. Both Compact myostatin and genetic background exhibit systemic metabolic effects. The Compact mutation decreases adiposity, improves whole body glucose uptake, insulin sensitivity and 18FDG uptake of skeletal muscle and white adipose tissue, while the Compact genetic background has opposite effect. Importantly, the mutation does not prevent the formation of mature myostatin; however, a decrease in myostatin level was observed leading to altered activation of Smad2, Smad1/5/8 and Akt, and increased level of pAS160, a Rab-GTPase activating protein responsible for GLUT4 translocation. Based on our analysis the Compact genetic background strengthens the effect of myostatin mutation on muscle mass, but can compensate each other when systemic metabolic effects are compared