Effect of rs4646994 polymorphism of angiotensin-converting enzyme on the risk of nonischemic cardiomyopathy.

BACKGROUND: Angiotensin-converting enzyme (ACE) gene polymorphisms have recently been shown to be associated with risk of developing left ventricular hypertrophy (LVH). However, the results were controversial. We aimed to conduct this meta-analysis to further confirm the association between ACE rs4646994 polymorphism and hypertrophic cardiomyopathy (HCM)/dilated cardiomyopathy (DCM). METHODS: PubMed, Embase, the Chinese National Knowledge Information, and Wanfang databases were searched for eligible studies. The Newcastle-Ottawa Scale (NOS) was used to evaluate the quality of included studies. Then we evaluated the association between ACE gene mutation and HCM/DCM by calculating odds ratios (ORs) and 95% confidence intervals (95% CIs). Subgroup analysis was further performed to explore situations in specialized subjects. Sensitivity analysis and publication bias was assessed to confirm the study reliability. RESULTS: There were 13 studies on DCM (2004 cases and 1376 controls) and 16 studies on HCM (2161 controls and 1192 patients). ACE rs4646994 polymorphism was significantly associated with DCM in all genetic models. However, in HCM, four genetic models (allele model, homozygous model, heterozygous model, and dominant model) showed significant association between ACE rs4646994 polymorphism and DCM. In subgroup analysis, we found that ACE rs4646994 polymorphism was significantly associated with DCM/HCM in Asian population. Finally, we also conducted a cumulative meta-analysis, which indicates that the results of our meta-analysis are highly reliable. CONCLUSION: ACE rs4646994 polymorphism increases the risk of DCM/HCM in Asians, but not in Caucasians. More case-control studies are needed to strengthen our conclusions and to assess the gene-gene and gene-environment interactions between ACE rs4646994 polymorphism and DCM/HCM.

Self-Maintenance of Cardiac Resident Reparative Macrophages Attenuates Doxorubicin-Induced Cardiomyopathy Through the SR-A1-c-Myc Axis.

RATIONALE: Doxorubicin-induced cardiomyopathy (DiCM) is a primary cause of heart failure and mortality in cancer patients, in which macrophage-orchestrated inflammation serves as an essential pathological mechanism. However, the specific roles of tissue-resident and monocyte-derived macrophages in DiCM remain poorly understood. OBJECTIVE: Uncovering the origins, phenotypes, and functions of proliferative cardiac resident macrophages and mechanistic insights into the self-maintenance of cardiac macrophage during DiCM progression. METHODS AND RESULTS: Mice were administrated with doxorubicin to induce cardiomyopathy. Dynamic changes of resident and monocyte-derived macrophages were examined by lineage tracing, parabiosis, and bone marrow transplantation. We found that the monocyte-derived macrophages primarily exhibited a proinflammatory phenotype that dominated the whole DiCM pathological process and impaired cardiac function. In contrast, cardiac resident macrophages were vulnerable to doxorubicin insult. The survived resident macrophages exhibited enhanced proliferation and conferred a reparative role. Global or myeloid specifically ablation of SR-A1 (class A1 scavenger receptor) inhibited proliferation of cardiac resident reparative macrophages and, therefore, exacerbated cardiomyopathy in DiCM mice. Importantly, the detrimental effect of macrophage SR-A1 deficiency was confirmed by transplantation of bone marrow. At the mechanistic level, we show that c-Myc (Avian myelocytomatosis virus oncogene cellular homolog), a key transcriptional factor for the SR-A1-P38-SIRT1 (Sirtuin 1) pathway, mediated the effect of SR-A1 in reparative macrophage proliferation in DiCM. CONCLUSIONS: The SR-A1-c-Myc axis may represent a promising target to treat DiCM through augmentation of cardiac resident reparative macrophage proliferation.

Up-regulating autophagy by targeting the mTOR-4EBP1 pathway: a possible mechanism for improving cardiac function in mice with experimental dilated cardiomyopathy.

BACKGROUND: Autophagy plays a crucial role in the pathological process of cardiovascular diseases. However, little is known about the pathological mechanism underlying autophagy regulation in dilated cardiomyopathy (DCM). METHODS: We explored whether up-regulating autophagy could improve cardiac function in mice with experimental DCM through the mTOR-4EBP1 pathway. Animal model of DCM was established in BALB/c mice by immunization with porcine cardiac myosin. Both up- or down-regulation of autophagy were studied by administration of rapamycin or 3-MA in parallel. Morphology, Western blotting, and echocardiography were applied to confirm the pathological mechanisms. RESULTS: Autophagy was activated and autophagosomes were significantly increased in the rapamycin group. The collagen volume fraction (CVF) was decreased in the rapamycin group compared with the DCM group (9.21 ± 0.82% vs 14.38 ± 1.24%, P < 0.01). The expression of p-mTOR and p-4EBP1 were significantly decreased in rapamycin-induced autophagy activation, while the levels were increased by down-regulating autophagy with 3-MA. In the rapamycin group, the LVEF and FS were significantly increased compared with the DCM group (54.12 ± 6.48% vs 45.29 ± 6.68%, P < 0.01; 26.89 ± 4.04% vs 22.17 ± 2.82%, P < 0.05). As the inhibitor of autophagy, 3-MA aggravated the progress of maladaptive cardiac remodeling and declined cardiac function in DCM mice. CONCLUSIONS: The study indicated a possible mechanism for improving cardiac function in mice with experimental DCM by up-regulating autophagy via the mTOR-4EBP1 pathway, which could be a promising therapeutic strategy for DCM.

Homozygous damaging SOD2 variant causes lethal neonatal dilated cardiomyopathy.

BACKGROUND: Idiopathic dilated cardiomyopathy (DCM) is recognised to be a heritable disorder, yet clinical genetic testing does not produce a diagnosis in >50% of paediatric patients. Identifying a genetic cause is crucial because this knowledge can affect management options, cardiac surveillance in relatives and reproductive decision-making. In this study, we sought to identify the underlying genetic defect in a patient born to consanguineous parents with rapidly progressive DCM that led to death in early infancy. METHODS AND RESULTS: Exome sequencing revealed a potentially pathogenic, homozygous missense variant, c.542G>T, p.(Gly181Val), in SOD2. This gene encodes superoxide dismutase 2 (SOD2) or manganese-superoxide dismutase, a mitochondrial matrix protein that scavenges oxygen radicals produced by oxidation-reduction and electron transport reactions occurring in mitochondria via conversion of superoxide anion (O2-·) into H2O2. Measurement of hydroethidine oxidation showed a significant increase in O2-· levels in the patient's skin fibroblasts, as compared with controls, and this was paralleled by reduced catalytic activity of SOD2 in patient fibroblasts and muscle. Lentiviral complementation experiments demonstrated that mitochondrial SOD2 activity could be completely restored on transduction with wild type SOD2. CONCLUSION: Our results provide evidence that defective SOD2 may lead to toxic increases in the levels of damaging oxygen radicals in the neonatal heart, which can result in rapidly developing heart failure and death. We propose SOD2 as a novel nuclear-encoded mitochondrial protein involved in severe human neonatal cardiomyopathy, thus expanding the wide range of genetic factors involved in paediatric cardiomyopathies.

Reversion of cardiac dysfunction by a novel orally available calcium/calmodulin-dependent protein kinase II inhibitor, RA306, in a genetic model of dilated cardiomyopathy.

AIMS: Despite improvements in patient identification and management, heart failure (HF) remains a major public health burden and an important clinical challenge. A variety of animal and human studies have provided evidence suggesting a central role of calcium/calmodulin-dependent protein kinase II (CaMKII) in the development of pathological cardiac remodelling and HF. Here, we describe a new potent, selective, and orally available CaMKII inhibitor. METHODS AND RESULTS: Chemical optimization led to the identification of RA306 as a selective CaMKII inhibitor. This compound was found potent on the cardiac CaMKII isoforms delta and gamma (IC50 in the 10 nM range), with pharmacokinetic properties allowing oral administration in animal models of HF. RA306 was administered to diseased mice carrying a mutation in alpha-actin that is responsible for dilated cardiomyopathy (DCM) in humans. In two separate studies, RA306 was orally administered at 30 mg/kg either for 2 weeks (twice a day) or for 2 months (once a day). Echocardiography monitoring showed that RA306 significantly improved cardiac function (ejection fraction and cardiac output) as compared to vehicle. These disease modifying effects of RA306 were associated with inhibition of cardiac phosphorylation of phospholamban (PLN) at threonine-17, indicating reduced cardiac CaMKII activity. CONCLUSION: This work supports the feasibility of identifying potent orally available CaMKII inhibitors suitable for clinical use to treat heart disease.

Immunoexpression of MMP-8, MMP-9 and TIMP-2 in dilated cardiomyopathy.

Alteration of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) expression has been studied for various cardiac diseases, including dilated cardiomyopathy (DCM), with the significance of surrogate markers of extracellular matrix (ECM) remodeling. In this study, we determined the MMP-8, MMP-9 and TIMP-2 immunoexpression in the heart of patients diagnosed with DCM in relation to a histological composite score (HCS). The study included 40 cases of heart fragments that were processed by the usual paraffin inclusion technique, followed by a semi-quantitative evaluation of histopathological parameters, which summed, allowed the establishment of a HCS. Subsequently, the cases were immunohistochemically processed for MMP-8, MMP-9 and TIMP-2, followed by the semi-quantitative evaluation of their expression intensity. MMP-8 was identified only in myocardiocytes, while MMP-9 and TIMP-2 were present in both myocardiocytes and stroma, but with different intensity. The increasing intensity of MMP-8 and TIMP-2 immunoreactions was significantly associated with low HCS. In case of MMP-9, the immunostaining intensity analysis in relation to the HCS level revealed insignificant differences, but we found an association of increased and moderate intensity with low HCS. The imbalance between TIMPs and MMPs disrupts the ECM architecture and contributes to the remodeling process in DCM, aspect that can be used in the development of new clinical therapies.

Phenotypic spectrum of ALPK3-related cardiomyopathy.

Cardiomyopathies are clinically heterogeneous disorders and are the leading cause of cardiovascular morbidity and mortality. Different etiologies have a significant impact on prognosis. Recently, novel biallelic loss-of-function pathogenic variants in alpha-kinase 3 (ALPK3) were implicated in causing early-onset pediatric cardiomyopathy (cardiomyopathy, familial hypertrophic 27; OMIM 618052). To date, eight patients, all presented during early childhood, were reported with biallelic ALPK3 pathogenic variants. We describe the molecular and clinical phenotype characterization of familial cardiomyopathy on one family with six affected individuals. We identified homozygosity for an ALPK3 deleterious sequence variant (NM_020778.4:c.639G>A:p.Trp213*) in all the affected individuals. They presented with either dilated cardiomyopathy that progressed to hypertrophic cardiomyopathy (HCM) or HCM with left ventricular noncompaction. The age of presentation in our cohort extends between infancy to the fourth decade. The phenotypic severity decreases with the progression of age.

ITPase deficiency causes a Martsolf-like syndrome with a lethal infantile dilated cardiomyopathy.

Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated "mutation negative" probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA-and by implication rI production-correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.

SPEG Controls Calcium Reuptake Into the Sarcoplasmic Reticulum Through Regulating SERCA2a by Its Second Kinase-Domain.

RATIONALE: SPEG (Striated muscle preferentially expressed protein kinase) has 2 kinase-domains and is critical for cardiac development and function. However, it is not clear how these 2 kinase-domains function to maintain cardiac performance. OBJECTIVE: To determine the molecular functions of the 2 kinase-domains of SPEG. METHODS AND RESULTS: A proteomics approach identified SERCA2a (sarcoplasmic/endoplasmic reticulum calcium ATPase 2a) as a protein interacting with the second kinase-domain but not the first kinase-domain of SPEG. Furthermore, the second kinase-domain of SPEG could phosphorylate Thr484 on SERCA2a, promote its oligomerization and increase calcium reuptake into the sarcoplasmic/endoplasmic reticulum in culture cells and primary neonatal rat cardiomyocytes. Phosphorylation of SERCA2a by SPEG enhanced its calcium-transporting activity without affecting its ATPase activity. Depletion of Speg in neonatal rat cardiomyocytes inhibited SERCA2a-Thr484 phosphorylation and sarcoplasmic reticulum calcium reuptake. Moreover, overexpression of SERCA2aThr484Ala mutant protein also slowed sarcoplasmic reticulum calcium reuptake in neonatal rat cardiomyocytes. In contrast, domain mapping and phosphorylation analysis revealed that the first kinase-domain of SPEG interacted and phosphorylated its recently identified substrate JPH2 (junctophilin-2). An inducible heart-specific Speg knockout mouse model was generated to further study this SPEG-SERCA2a signal nexus in vivo. Inducible deletion of Speg decreased SERCA2a-Thr484 phosphorylation and its oligomerization in the heart. Importantly, inducible deletion of Speg inhibited SERCA2a calcium-transporting activity and impaired calcium reuptake into the sarcoplasmic reticulum in cardiomyocytes, which preceded morphological and functional alterations of the heart and eventually led to heart failure in adult mice. CONCLUSIONS: Our data demonstrate that the 2 kinase-domains of SPEG may play distinct roles to regulate cardiac function. The second kinase-domain of SPEG is a critical regulator for SERCA2a. Our findings suggest that SPEG may serve as a new target to modulate SERCA2a activation for treatment of heart diseases with impaired calcium homeostasis.

(-)-Epicatechin inhibits development of dilated cardiomyopathy in δ sarcoglycan null mouse.

BACKGROUND AND AIMS: Several studies propose that (-)-epicatechin, a flavonol present in high concentration in the cocoa, has cardioprotective effects. This study aimed to evaluate the impact of (-)-epicatechin on the development of dilated cardiomyopathy in a δ sarcoglycan null mouse model. METHODS AND RESULTS: δ Sarcoglycan null mice were treated for 15 days with (-)-epicatechin. Histological and morphometric analysis of the hearts treated mutant mice showed significant reduction of the vasoconstrictions in the coronary arteries as well as fewer areas with fibrosis and a reduction in the loss of the ventricular wall. On the contrary, it was observed a thickening of this region. By Western blot analysis, it was shown, and increment in the phosphorylation level of eNOS and PI3K/AKT/mTOR/p70S6K proteins in the heart of the (-)-epicatechin treated animals. On the other hand, we observed a significantly decreased level of the atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) heart failure markers. CONCLUSION: All the results indicate that (-)-epicatechin has the potential to prevent the development of dilated cardiomyopathy of genetic origin and encourages the use of this flavonol as a pharmacological therapy for dilated cardiomyopathy and heart failure diseases.

Mutations in PPCS, Encoding Phosphopantothenoylcysteine Synthetase, Cause Autosomal-Recessive Dilated Cardiomyopathy.

Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.

A Disintegrin and Metalloprotease-22 Attenuates Hypertrophic Remodeling in Mice Through Inhibition of the Protein Kinase B Signaling Pathway.

BACKGROUND: Severe cardiac hypertrophy can lead to cardiac remodeling and even heart failure in the end, which is a leading cause of cardiovascular disease-related mortality worldwide. A disintegrin and metalloprotease-22 (ADAM22), a member of the transmembrane and secreted metalloendopeptidase family, participates in many biological processes, including those in the cardiovascular system. However, there is no explicit information on whether ADAM22 can regulate the process of cardiac hypertrophy; the effects that ADAM22 exerts in cardiac hypertrophy remain elusive. METHODS AND RESULTS: We observed significantly increased ADAM22 expression in failing hearts from patients with dilated cardiomyopathy and hypertrophic cardiomyopathy; the same trend was observed in mice induced by transaortic constriction and in neonatal rat cardiomyocytes treated by angiotensin II. Therefore, we constructed both cardiac-specific ADAM22 overexpression and knockout mice. At 4 weeks after transaortic constriction, cardiac-specific ADAM22 knockout, by the CRISPR/Cas9 (clustered regularly interspaced palindromic repeat (CRISPR)-Cas9) system, deteriorated the severity of cardiac hypertrophy in mice, whereas cardiac-specific ADAM22 overexpression mitigated the degrees of cardiac hypertrophy in mice. Similarly, altered ADAM22 expression modulated the angiotensin II-mediated cardiomyocyte hypertrophy in neonatal rat cardiomyocytes. After screening several signaling pathways, we found ADAM22 played a role in inhibition of protein kinase B (AKT) activation. Under the cardiac-specific ADAM22 knockout background, AKT activation was enhanced in transaortic constriction-induced mice and angiotensin II-stimulated neonatal rat cardiomyocytes, with a severe degree of cardiac hypertrophy. Treatment of a specific AKT inhibitor attenuated the transaortic constriction-enhanced AKT activation and cardiac hypertrophy in mice. CONCLUSIONS: The findings demonstrated that ADAM22 negatively regulates the AKT activation and the process of cardiac hypertrophy and may provide new insights into the pathobiological features of cardiac hypertrophy.

Serum paraoxonase activity in patients with ischaemic and nonischaemic dilated cardiomyopathy.

BACKGROUND: This study examined whether the serum PON1 activity is different in patients with ischaemic dilated cardiomyopathy (IDCM) and nonischaemic dilated cardiomyopathy (NDCM) and the relation between the serum PON1 activity and serum pro-BNP levels. METHODS AND RESULTS: In this study, we enrolled 60 patients with left ventricular systolic failure (New York Heart Association [NYHA] class III-IV) and a left ventricular ejection fraction (EF) < 40% as determined by echocardiography and 30 healthy subjects. The patients with systolic heart failure were divided into two groups: patients with IDCM and patients with NDCM. Blood samples were obtained to measure the serum PON1 activity and the serum pro-BNP levels. The median serum PON1 activities were lower among the patients with IDCM or with NDCM compared with the control subjects (p < .001, p = .043, respectively). Compared with the control subjects, the patients with IDCM or with NDCM had higher serum pro-BNP levels (p < .001, p < .001, respectively). The serum PON1 activity was negatively correlated with the serum pro-BNP levels in patients with IDCM (r = -0.548, p < .001). The area under the ROC curve of the serum PON1 activity was 0.798. Using a serum PON1 activity of 201.3 U/L as a cut-off value, the sensitivity was 86.84% and specificity was 66.67% for the diagnosis of IDCM. CONCLUSIONS: In this study, the serum PON1 activity was significantly reduced in the patients with IDCM or with NDCM compared with the control subjects. The serum PON1 activity of the patients with IDCM was negatively correlated with the serum pro-BNP levels.

Identification of MYLK3 mutations in familial dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a primary cause of heart failure, life-threatening arrhythmias, and cardiac death. Pathogenic mutations have been identified at the loci of more than 50 genes in approximately 50% of DCM cases, while the etiologies of the remainder have yet to be determined. In this study, we applied whole exome sequencing in combination with segregation analysis to one pedigree with familial DCM, and identified a read-through mutation (c.2459 A > C; p.*820Sext*19) in the myosin light chain kinase 3 gene (MYLK3). We then conducted MYLK3 gene screening of 15 DCM patients (7 familial and 8 sporadic) who were negative for mutation screening of the previously-reported cardiomyopathy-causing genes, and identified another case with a MYLK3 frameshift mutation (c.1879_1885del; p.L627fs*41). In vitro experiments and immunohistochemistry suggested that the MYLK3 mutations identified in this study result in markedly reduced levels of protein expression and myosin light chain 2 phosphorylation. This is the first report that MYLK3 mutations can cause DCM in humans. The clinical phenotypes of DCM patients were consistent with MYLK3 loss-of-function mouse and zebrafish models in which cardiac enlargement and heart failure are observed. Our findings highlight an essential role for cardiac myosin light chain kinase in the human heart.

Impaired Protein Quality Control During Left Ventricular Remodeling in Mice With Cardiac Restricted Overexpression of Tumor Necrosis Factor.

BACKGROUND: Sustained inflammation in the heart is sufficient to provoke left ventricular dysfunction and left ventricular remodeling. Although inflammation has been linked to many of the biological changes responsible for adverse left ventricular remodeling, the relationship between inflammation and protein quality control in the heart is not well understood. METHODS AND RESULTS: To study the relationship between chronic inflammation and protein quality control, we used a mouse model of dilated cardiomyopathy driven by cardiac restricted overexpression of TNF (tumor necrosis factor; Myh6-sTNF). Myh6-sTNF mice develop protein aggregates containing ubiquitin-tagged proteins within cardiac myocytes related to proteasome dysfunction and impaired autophagy. The 26S proteasome was dysfunctional despite normal function of the core 20S subunit. We found an accumulation of autophagy substrates in Myh6-sTNF mice, which were also seen in tissue from patients with end-stage heart failure. Moreover, there was evidence of impaired autophagosome clearance after chloroquine administration in these mice indicative of impaired autophagic flux. Finally, there was increased mammalian target of rapamycin complex 1 (mTORC1) activation, which has been linked to inhibition of both the proteasome and autophagy. CONCLUSIONS: Myh6-sTNF mice with sustained inflammatory signaling develop proteasome dysfunction and impaired autophagic flux that is associated with enhanced mTORC1 activation.

Inhibition of mevalonate pathway prevents ischemia-induced cardiac dysfunction in rats via RhoA-independent signaling pathway.

AIM: We previously demonstrated that anoxia-mediated Ca2+ handling dysfunction could be ameliorated through inhibition of mevalonate pathway via RhoA- and Ras-related mechanisms in H9c2 cells. In this study, we further explored whether inhibition of mevalonate pathway is associated with cardiac remodeling and dysfunction in ischemic cardiomyopathy, and discussed the possible role of Ras, Rac and RhoA in cardiac dysfunction. METHODS: We investigated the role of mevalonate pathway in cardiac remodeling and cardiomyocyte Ca2+ handling proteins expression in a rat model of cardiac dysfunction due to myocardial infarction (MI). After MI, adult male Sprague-Dawley rats were treated with drugs that antagonize key components in mevalonate pathway, including 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, and Rho-kinase for 10 weeks. The protein expression of ryanodine receptor 2 (RyR2), sarcoplasmic reticulum Ca2+ ATPase (SERCA) 2a, phospholamban (PLB), phospho-PLB at serine-16 (PSer16-PLB), FKBP12.6, and RhoA as well as RyR2 and FKBP12.6 mRNA levels was evaluated. RESULTS: Rosuvastatin and alendronate treatment prevented myocardial remodeling, improved cardiac function and reduced infarct size. Furthermore, rosuvastatin and alendronate promoted an increase in the protein expression of SERCA2a and PSer16-PLB/PLB ratio as well as partially restored the RyR2 and FKBP12.6 gene and protein expression. Fasudil failed to exert these beneficial effects. CONCLUSIONS: These findings indicate that mevalonate pathway inhibition by rosuvastatin and alendronate prevents cardiac remodeling and dysfunction possibly through RhoA-independent mechanisms.

Opposite effects of catalase and MnSOD ectopic expression on stress induced defects and mortality in the desmin deficient cardiomyopathy model.

Oxidative stress has been linked strongly to cell death and cardiac remodeling processes, all hallmarks of heart failure. Mice deficient for desmin (des-/-), the major muscle specific intermediate filament protein, develop dilated cardiomyopathy and heart failure characterized by mitochondrial defects and cardiomyocyte death. The cellular and biochemical alterations in the hearts of these mice strongly suggest that oxidative stress is one of the mechanisms contributing to the pathogenesis of the phenotype. Recently, we showed that indeed the desmin deficient cardiomyocytes are under increased oxidative stress. In order to verify these findings in vivo, we generated transgenic animals overexpressing SOD2 (MnSOD) and/or catalase in the heart and crossed them with des-/- mice, thus allowing us to evaluate the contribution of oxidative injury in inherited cardiomyopathies, as well as the therapeutic potential of antioxidant strategies. Moderate MnSOD and/or catalase overexpression in des-/- hearts leads to a marked decrease in intracellular reactive oxygen species (ROS), ameliorates mitochondrial and other ultrastructural defects, minimizes myocardial degeneration and leads to a significant improvement of cardiac function. Importantly, catalase overexpression increased the 50% survival rate of des-/- mice in an obligatory exercise to 100%. In contrast, MnSOD overexpression enhanced the lethality of des-/- mice, underscoring the importance of a fine balanced cellular redox status. Overall, the present study supports the contribution of oxidative stress in the development of des-/- cardiomyopathy and points to a well-considered antioxidant treatment as therapeutic for cardiomyopathies.

The E3 ubiquitin ligase c-Cbl mediates integrin ß1 ubiquitination during dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by dilatation of the ventricular chambers and impaired myocardial contractility. The results of our previous study indicated that a deficiency in matricellular cartilage oligomeric matrix protein (COMP) led to spontaneous and progressive DCM in mice via the ubiquitination/degradation of integrin ß1. However, the specific ubiquitin enzyme involved in degradation of integrin ß1 and the pathogenesis of DCM remain elusive. We first compared gene expression profiles in hearts from 3-month-old wild type and COMP-/- mice using microarray analysis. Among the E3 ubiquitin ligases upregulated in COMP-/- hearts, c-Cbl silencing rescued the ubiquitination/degradation of integrin ß1, myofilament loss, apoptosis and connexin-43 deficiency in cardiomyocytes due to the silencing of COMP. Furthermore, c-Cbl silencing by intramyocardial injections of siRNA into 1-month-old COMP-/- mice ameliorated spontaneous DCM in vivo, as evidenced by the inhibition of the dilation of ventricular chambers, impaired ejection fraction and myofilament loss. A subsequent cellular ubiquitination assay revealed that overexpression of c-Cbl induced ubiquitination of integrin ß1, whereas the G306E mutation in c-Cbl, which prevented the binding of c-Cbl to its substrates, had no effect on integrin ß1 ubiquitination, indicating that c-Cbl directly caused the ubiquitination of integrin ß1 in the hearts. In conclusion, our results demonstrate that c-Cbl mediates the ubiquitination/degradation of integrin ß1, which leads to COMP deficiency-induced DCM.

Dyrk1a regulates the cardiomyocyte cell cycle via D-cyclin-dependent Rb/E2f-signalling.

AIMS: Down syndrome-associated dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A) is a ubiquitously expressed protein kinase. Up to date a variety of targets have been identified, establishing a key role for Dyrk1a in selected signalling pathways. In cardiomyocytes, Dyrk1a acts as a negative regulator of hypertrophy by phosphorylating transcription factors of the NFAT family, but its mechanistic function in the heart remains poorly understood. This study was designed to investigate a potential protective role of Dyrk1a in cardiac hypertrophy in vivo. METHODS AND RESULTS: We generated transgenic mice with cardiac-specific overexpression of Dyrk1a. Counterintuitively, these mice developed severe dilated cardiomyopathy associated with congestive heart failure and premature death. In search for the cause of this unexpected phenotype, we found that Dyrk1a interacts with all members of the D-cyclin family and represses their protein levels in vitro and in vivo. Particularly, forced expression of Dyrk1a leads to increased phosphorylation of Ccnd2 on Thr280 and promotes its subsequent proteasomal degradation. Accordingly, cardiomyocytes overexpressing Dyrk1a display hypo-phosphorylated Rb1, suppression of Rb/E2f-signalling, and reduced expression of E2f-target genes, which ultimately results in impaired cell cycle progression. CONCLUSIONS: We identified Dyrk1a as a novel negative regulator of D-cyclin-mediated Rb/E2f-signalling. As dysregulation of this pathway with impaired cardiomyocyte proliferation leads to cardiomyopathy, dose-specific Dyrk1a expression and activity appears to be critical for the hyperplastic and hypertrophic growth of the developing heart.

FasL expression in cardiomyocytes activates the ERK1/2 pathway, leading to dilated cardiomyopathy and advanced heart failure.

Increase in the apoptotic molecule Fas ligand (FasL) in serum and cardiomyocytes has been shown to be associated with progressive dilated cardiomyopathy (DCM) and congestive heart failure (CHF) in humans. However, the underlying mechanism(s) of FasL-related deterioration of heart function remain obscure. The aim of the present study is to determine roles of myocardial FasL in the activation of alternative pathways such as extracellular-signal-regulated kinase 1/2 (ERK1/2), inflammation or fibrosis and to identify effective treatments of progressive DCM and advanced CHF. Transgenic mice with cardiomyocyte-specific overexpression of FasL were investigated and treated with an ERK1/2 inhibitor (U-0126), losartan (los), prednisolone (pred) or placebo. Morpho-histological and molecular studies were subsequently performed. FasL mice showed significantly higher mortality compared with wild-type (WT) littermates due to DCM and advanced CHF. Prominent perivascular and interstitial fibrosis, increased interleukin secretion and diffuse CD3-positive cell infiltration were evident in FasL hearts. Up-regulation of the short form of Fas-associated death domain (FADD)-like interleukin 1ß-converting enzyme (FLICE) inhibitory protein (s-FLIP), RIP (receptor-interacting protein) and ERK1/2 and down-regulation of transforming growth factor beta 1 (TGFß1) and nuclear factor-κB (NF-κB) was determined in the myocardium, whereas expression of ERK1/2, periostin (Postn) and osteopontin increased in cardiac fibroblasts. U-0126 and los increased CHF survival by 75% compared with pred and placebo groups. U-0126 had both anti-fibrotic and anti-apoptotic effects, whereas los reduced fibrosis only. Myocardial FasL expression in mice activates differential robust fibrotic, apoptotic and inflammatory responses via ERK1/2 in cardiomyocytes and cardiac fibroblasts inducing DCM and CHF. Blocking the ERK1/2 pathway prevented progression of FasL-induced DCM and CHF by reducing fibrosis, inflammation and apoptosis in the myocardium.