Unraveling Desmin's Head Domain Structure and Function.

Understanding the structure and function of intermediate filaments (IFs) is necessary in order to explain why more than 70 related IF genes have evolved in vertebrates while maintaining such dramatically tissue-specific expression. Desmin is a member of the large multigene family of IF proteins and is specifically expressed in myocytes. In an effort to elucidate its muscle-specific behavior, we have used a yeast two-hybrid system in order to identify desmin's head binding partners. We described a mitochondrial and a lysosomal protein, NADH ubiquinone oxidoreductase core subunit S2 (NDUFS2), and saposin D, respectively, as direct desmin binding partners. In silico analysis indicated that both interactions at the atomic level occur in a very similar way, by the formation of a three-helix bundle with hydrophobic interactions in the interdomain space and hydrogen bonds at R16 and S32 of the desmin head domain. The interactions, confirmed also by GST pull-down assays, indicating the necessity of the desmin head domain and, furthermore, point out its role in function of mitochondria and lysosomes, organelles which are disrupted in myopathies due to desmin head domain mutations.

Myospryn deficiency leads to impaired cardiac structure and function and schizophrenia-associated symptoms.

The desmin-associated protein myospryn, encoded by the cardiomyopathy-associated gene 5 (CMYA5), is a TRIM-like protein associated to the BLOC-1 (Biogenesis of Lysosomes Related Organelles Complex 1) protein dysbindin. Human myospryn mutations are linked to both cardiomyopathy and schizophrenia; however, there is no evidence of a direct causative link of myospryn to these diseases. Therefore, we sought to unveil the role of myospryn in heart and brain. We have genetically inactivated the myospryn gene by homologous recombination and demonstrated that myospryn null hearts have dilated phenotype and compromised cardiac function. Ultrastructural analyses revealed that the sarcomere organization is not obviously affected; however, intercalated disk (ID) integrity is impaired, along with mislocalization of ID and sarcoplasmic reticulum (SR) protein components. Importantly, cardiac and skeletal muscles of myospryn null mice have severe mitochondrial defects with abnormal internal vacuoles and extensive cristolysis. In addition, swollen SR and T-tubules often accompany the mitochondrial defects, strongly implying a potential link of myospryn together with desmin to SR- mitochondrial physical and functional cross-talk. Furthermore, given the reported link of human myospryn mutations to schizophrenia, we performed behavioral studies, which demonstrated that myospryn-deficient male mice display disrupted startle reactivity and prepulse inhibition, asocial behavior, decreased exploratory behavior, and anhedonia. Brain neurochemical and ultrastructural analyses revealed prefrontal-striatal monoaminergic neurotransmitter defects and ultrastructural degenerative aberrations in cerebellar cytoarchitecture, respectively, in myospryn-deficient mice. In conclusion, myospryn is essential for both cardiac and brain structure and function and its deficiency leads to cardiomyopathy and schizophrenia-associated symptoms.

Crosstalk between coagulation and complement activation promotes cardiac dysfunction in arrhythmogenic right ventricular cardiomyopathy.

Aims: We previously found that complement components are upregulated in the myocardium of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), and inhibiting the complement receptor C5aR reduces disease severity in desmin knockout (Des-/- ) mice, a model for ARVC. Here, we examined the mechanism underlying complement activation in ARVC, revealing a potential new therapeutic target. Methods: First, immunostaining, RT-PCR and western blot were used to detect the expression levels of complement and coagulation factors. Second, we knocked out the central complement component C3 in Des-/- mice (ARVC model) by crossing Des-/- mice with C3-/- mice to explore whether complement system activation occurs independently of the conventional pathway. Then, we evaluated whether a targeted intervention to coagulation system is effective to reduce myocardium injury. Finally, the plasma sC5b9 level was assessed to investigate the role in predicting adverse cardiac events in the ARVC cohort. Results: The complement system is activated in the myocardium in ARVC. Autoantibodies against myocardial proteins provided a possible mechanism underlying. Moreover, we found increased levels of myocardial C5 and the serum C5a in Des-/-C3-/- mice compared to wild-type mice, indicating that C5 is activated independently from the conventional pathway, presumably via the coagulation system. Crosstalk between the complement and coagulation systems exacerbated the myocardial injury in ARVC mice, and this injury was reduced by using the thrombin inhibitor lepirudin. In addition, we found significantly elevated plasma levels of sC5b9 and thrombin in patients, and this increase was correlated with all-cause mortality. Conclusions: These results suggest that crosstalk between the coagulation and complement systems plays a pathogenic role in cardiac dysfunction in ARVC. Thus, understanding this crosstalk may have important clinical implications with respect to diagnosing and treating ARVC.

Desmin is essential for the structure and function of the sinoatrial node: implications for increased arrhythmogenesis.

Our objective was to investigate the effect of desmin depletion on the structure and function of the sinoatrial pacemaker complex (SANcl) and its implication in arrhythmogenesis. Analysis of mice and humans (SANcl) indicated that the sinoatrial node exhibits high amounts of desmin, desmoplakin, N-cadherin, and ß-catenin in structures we call "lateral intercalated disks" connecting myocytes side by side. Examination of the SANcl from an arrhythmogenic cardiomyopathy model, desmin-deficient (Des-/-) mouse, by immunofluorescence, ultrastructural, and Western blot analysis showed that the number of these lateral intercalated disks was diminished. Also, electrophysiological recordings of the isolated compact sinoatrial node revealed increased pacemaker systolic potential and higher diastolic depolarization rate compared with wild-type mice. Prolonged interatrial conduction expressed as a longer P wave duration was also observed in Des-/- mice. Upregulation of mRNA levels of both T-type Ca2+ current channels, Cav3.1 and Cav3.2, in the Des-/- myocardium (1.8- and 2.3-fold, respectively) and a 1.9-fold reduction of funny hyperpolarization-activated cyclic nucleotide-gated K+ channel 1 could underlie these functional differences. To investigate arrhythmogenicity, electrocardiographic analysis of Des-deficient mice revealed a major increase in supraventricular and ventricular ectopic beats compared with wild-type mice. Heart rate variability analysis indicated a sympathetic predominance in Des-/- mice, which may further contribute to arrhythmogenicity. In conclusion, our results indicate that desmin elimination leads to structural and functional abnormalities of the SANcl. These alterations may be enhanced by the sympathetic component of the cardiac autonomic nervous system, which is predominant in the desmin-deficient heart, thus leading to increased arrhythmogenesis.NEW & NOTEWORTHY The sinoatrial node exhibits high amounts of desmin and desmoplakin in structures we call "lateral intercalated disks," connecting side-by-side adjacent cardiomyocytes. These structures are diminished in desmin-deficient mouse models. Misregulation of T-type Ca2+ current and hyperpolarization-activated cyclic nucleotide-gated K+ channel 1 was proved along with prolonged interatrial conduction and cardiac autonomic nervous system dysfunction.

Amelioration of desmin network defects by αB-crystallin overexpression confers cardioprotection in a mouse model of dilated cardiomyopathy caused by LMNA gene mutation.

The link between the cytoplasmic desmin intermediate filaments and those of nuclear lamins serves as a major integrator point for the intracellular communication between the nucleus and the cytoplasm in cardiac muscle. We investigated the involvement of desmin in the cardiomyopathy caused by the lamin A/C gene mutation using the LmnaH222P/H222P mouse model of the disease. We demonstrate that in these mouse hearts desmin loses its normal Z disk and intercalated disc localization and presents aggregate formation along with mislocalization of basic intercalated disc protein components, as well as severe structural abnormalities of the intercalated discs and mitochondria. To address the extent by which the observed desmin network defects contribute to the progression of LmnaH222P/H222P cardiomyopathy, we investigated the consequences of desmin-targeted approaches for the disease treatment. We showed that cardiac-specific overexpression of the small heat shock protein αΒ-Crystallin confers cardioprotection in LmnaH222P/H222P mice by ameliorating desmin network defects and by attenuating the desmin-dependent mislocalization of basic intercalated disc protein components. In addition, αΒ-Crystallin overexpression rescues the intercalated disc, mitochondrial and nuclear defects of LmnaH222P/H222P hearts, as well as the abnormal activation of ERK1/2. Consistent with that, by generating the LmnaH222P/H222PDes+/- mice, we showed that the genetically decreased endogenous desmin levels have cardioprotective effects in LmnaH222P/H222P hearts since less desmin is available to form dysfunctional aggregates. In conclusion, our results demonstrate that desmin network disruption, disorganization of intercalated discs and mitochondrial defects are a major mechanism contributing to the progression of this LMNA cardiomyopathy and can be ameliorated by αΒ-Crystallin overexpression.

Complement system modulation as a target for treatment of arrhythmogenic cardiomyopathy.

Inflammation may contribute to disease progression in arrhythmogenic cardiomyopathy (ACM). However, its role in this process is unresolved. Our goal was to delineate the pathogenic role of the complement system in a new animal model of ACM and in human disease. Using cardiac histology, echocardiography, and electrocardiography, we have demonstrated that the desmin-null mouse (Des-/-) recapitulates most of the pathognomonic features of human ACM. Massive complement activation was observed in the Des-/- myocardium in areas of necrotic cells debris and inflammatory infiltrate. Analysis of C5aR-/-Des-/- double-null animals and a pharmaceutical approach using a C5a inhibitor were used to delineate the pathogenic role of the complement system in the disease progression. Our findings indicate that inhibiting C5aR (CD88) signaling improves cardiac function, histopathology, arrhythmias, and survival after endurance. Containment of the inflammatory reaction at the initiation of cardiac tissue injury (2-3 weeks of age), with consequently reduced myocardial remodeling and the absence of a direct long-lasting detrimental effect of C5a-C5aR signaling on cardiomyocytes, could explain the beneficial action of C5aR ablation in Des-/- cardiomyopathy. We extend the relevance of these findings to human pathophysiology by showing for the first time significant complement activation in the cardiac tissues of patients with ACM, thus suggesting that complement modulation could be a new therapeutic target for ACM.

A missense mutation in desmin tail domain linked to human dilated cardiomyopathy promotes cleavage of the head domain and abolishes its Z-disc localization.

A missense mutation (Ile 451 to Met) at the tail domain of the muscle-specific intermediate filament protein desmin has been suggested to be a genetic cause of dilated cardiomyopathy. The Ile451Met mutation is located inside a conserved motif in the desmin tail domain, believed to have a potential role in the lateral packing of type III intermediate filaments. Nevertheless, the role of the type III intermediate filament tail domain remains elusive. To further study the role of this domain in the function of cardiomyocytes and in the development of cardiomyopathy, we generated transgenic mice expressing the mutant desmin(I451M) in the cardiac tissue. Analysis of hearts from transgenic animals revealed that mutant desmin loses its Z-disc localization but it can still associate with the intercalated discs, which, however, have an altered architecture, resembling other examples of dilated cardiomyopathy. This is the first report demonstrating a critical role of the desmin head and tail domains in the formation of the IF scaffold around Z discs. It is further suggested that in cardiomyocytes, an interplay between desmin tail and head domains is taking place, which potentially protects the amino terminus of desmin from specific proteases. The fact that the association with intercalated discs seems unchanged suggests that this association must take place through the desmin tail, in contrast to the head domain that is most possibly involved in the Z-disc binding.

Eradication of Helicobacter bilis and H. hepaticus from infected mice by using a medicated diet.

Infection of laboratory mice with Helicobacter spp. is a serious problem for many laboratory animal facilities worldwide. Rederivation and antibiotic treatment are two of the most common methods used to eliminate the bacterial infection from rodent colonies. Forty-seven newly imported mice were suspected to be positive for Helicobacter infection based on PCR analysis of pooled fecal samples from sentinel animals. We treated the mice with a medicated feed containing four antibiotic compounds (amoxicillin, clarithromycin, metronidazole, omeprazole). After eight weeks of continuous administration the animals were negative for H. bilis and H. hepaticus. Frequent retesting of the animals for up to one year proved that the mouse colony remained negative for Helicobacter spp.