Knockdown of the TRPM4 channel alters cardiac electrophysiology and hemodynamics in a sex- and age-dependent manner in mice.

TRPM4 is a calcium-activated, voltage-modulated, nonselective ion channel widely expressed in various cells and tissues. TRPM4 regulates the influx of sodium ions, thus playing a role in regulating the membrane potential. In the heart, TRPM4 is expressed in both cardiomyocytes and cells of the conductive pathways. Clinical studies have linked TRPM4 mutations to several cardiac disorders. While data from experimental studies have demonstrated TRPM4's functional significance in cardiac physiology, its exact roles in the heart have remained unclear. In this study, we investigated the role of TRPM4 in cardiac physiology in a newly generated Trpm4 knockdown mouse model. Male and female Trpm4 knockdown (Trpm4-/- ) and wild-type mice of different ages (5- to 12- week-old (young) and 24-week-old or more (adult)) were characterized using a multimodal approach, encompassing surface electrocardiograms (ECG), echocardiography recordings, ex vivo ECGs in isolated heart, endocardial mappings, Western blots, and mRNA quantifications. The assessment of cardiac electrophysiology by surface ECGs revealed no significant differences between wild-type and Trpm4-/- young (5- to 12-week-old) mice of either sex. Above 24 weeks of age, adult male Trpm4-/- mice showed reduced heart rate and increased heart rate variability. Echocardiography revealed that only adult male Trpm4-/- mice exhibited slight left ventricular hypertrophic alterations compared to controls, illustrated by alterations of the mitral valve pressure halftime, the mitral valve E/A ratio, the isovolumetric relaxation time, and the mitral valve deceleration. In addition, an assessment of the right ventricular systolic function by scanning the pulmonary valve highlighted an alteration in pulmonary valve peak velocity and pressure in adult male Trpm4-/- mice. Endocardial mapping recordings showed that applying 5 µM of the new TRPM4 inhibitor NBA triggered a third-degree atrioventricular block on 40% of wild-type hearts. These results confirm the key role of TRPM4 in the proper structure and electrical function of the heart. It also reveals differences between male and female animals that have never been reported. In addition, the investigation of the effects of NBA on heart function confirms the role of TRPM4 in atrioventricular conduction.

Bioorthogonal site-selective conjugation of fluorescent dyes to antibodies: method and potential applications.

Antibodies are immensely useful tools for biochemical research and have found application in numerous protein detection and purification methods. Moreover, monoclonal antibodies are increasingly utilised as therapeutics or, conjugated to active pharmaceutical ingredients, in targeted chemotherapy. Several reagents and protocols are reported to synthesise fluorescent antibodies for protein target detection and immunofluorescence applications. However, most of these protocols lead to non-selective conjugation, over-labelling or in the worst case antigen binding site modification. Here, we have used the antibody disulphide cleavage and re-bridging strategy to introduce bright fluorescent dyes without loss of the antibody function. The resulting fluorescent IgG1 type antibodies were shown to be effective imaging tools in western blot and direct immunofluorescence experiments.

Deletion of Trpm4 Alters the Function of the Nav1.5 Channel in Murine Cardiac Myocytes.

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca2+-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na+ currents in myocytes. Hearts from Trpm4-/- mice presented increased sensitivity towards mexiletine, a Na+ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na+ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na+ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Nav1.5 function in murine cardiac myocytes.

Dystrophin and calcium current are decreased in cardiomyocytes expressing Cre enzyme driven by αMHC but not TNT promoter.

The Cre/lox system is a potent technology to control gene expression in mouse tissues. However, cardiac-specific Cre recombinase expression alone can lead to cardiac alterations when no loxP sites are present, which is not well understood. Many loxP-like sites have been identified in the mouse genome that might be Cre sensitive. One of them is located in the Dmd gene encoding dystrophin, a protein important for the function and stabilization of voltage-gated calcium (Cav1.2) and sodium (Nav1.5) channels, respectively. Here, we investigate whether Cre affects dystrophin expression and function in hearts without loxP sites in the genome. In mice expressing Cre under the alpha-myosin heavy chain (MHC-Cre) or Troponin T (TNT-Cre) promoter, we investigated dystrophin expression, Nav1.5 expression, and Cav1.2 function. Compared to age-matched MHC-Cre- mice, dystrophin protein level was significantly decreased in hearts from MHC-Cre+ mice of more than 12-weeks-old. Quantitative RT-PCR revealed decreased mRNA levels of Dmd gene. Unexpectedly, calcium current (ICaL), but not Nav1.5 protein expression was altered in those mice. Surprisingly, in hearts from 12-week-old and older TNT-Cre+ mice, neither ICaL nor dystrophin and Nav1.5 protein content were altered compared to TNT-Cre-. Cre recombinase unpredictably affects cardiac phenotype, and Cre-expressing mouse models should be carefully investigated before experimental use.

A Distinct Pool of Nav1.5 Channels at the Lateral Membrane of Murine Ventricular Cardiomyocytes.

Background: In cardiac ventricular muscle cells, the presence of voltage-gated sodium channels Nav1.5 at the lateral membrane depends in part on the interaction between the dystrophin-syntrophin complex and the Nav1.5 C-terminal PDZ-domain-binding sequence Ser-Ile-Val (SIV motif). α1-Syntrophin, a PDZ-domain adaptor protein, mediates the interaction between Nav1.5 and dystrophin at the lateral membrane of cardiac cells. Using the cell-attached patch-clamp approach on cardiomyocytes expressing Nav1.5 in which the SIV motif is deleted (ΔSIV), sodium current (INa) recordings from the lateral membrane revealed a SIV-motif-independent INa. Since immunostaining has suggested that Nav1.5 is expressed in transverse (T-) tubules, this remaining INa might be carried by channels in the T-tubules. Of note, a recent study using heterologous expression systems showed that α1-syntrophin also interacts with the Nav1.5 N-terminus, which may explain the SIV-motif independent INa at the lateral membrane of cardiomyocytes. Aim: To address the role of α1-syntrophin in regulating the INa at the lateral membrane of cardiac cells. Methods and Results: Patch-clamp experiments in cell-attached configuration were performed on the lateral membranes of wild-type, α1-syntrophin knockdown, and ΔSIV ventricular mouse cardiomyocytes. Compared to wild-type, a reduction of the lateral INa was observed in myocytes from α1-syntrophin knockdown hearts. Similar to ΔSIV myocytes, a remaining INa was still recorded. In addition, cell-attached INa recordings from lateral membrane did not differ significantly between non-detubulated and detubulated ΔSIV cardiomyocytes. Lastly, we obtained evidence suggesting that cell-attached patch-clamp experiments on the lateral membrane cannot record currents carried by channels in T-tubules such as calcium channels. Conclusion: Altogether, these results suggest the presence of a sub-pool of sodium channels at the lateral membrane of cardiomyocytes that is independent of α1-syntrophin and the PDZ-binding motif of Nav1.5, located in membrane domains outside of T-tubules. The question of a T-tubular pool of Nav1.5 channels, however, remains open.

PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function.

BACKGROUND: Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. METHODS AND RESULTS: To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (ΔSIV). ΔSIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of ΔSIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. CONCLUSIONS: Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Both complement and IgG fc receptors are required for development of attenuated antiglomerular basement membrane nephritis in mice.

To elucidate the mechanisms of glomerulonephritis, including Goodpasture's syndrome, mouse models are used that use heterologous Abs against the glomerular basement membrane (GBM) with or without preimmunization with foreign IgG from the same species. These studies have revealed the requirement of either FcgammaR or complement, depending on the experimental model used. In this study, we provide evidence that both FcgammaR and complement are obligatory for a full-blown inflammation in a novel attenuated passive model of anti-GBM disease. We demonstrate that administration of subnephritogenic doses of rabbit anti-GBM Abs followed by a fixed dose of mouse mAbs to rabbit IgG, allowing timing and dosing for the induction of glomerulonephritis, resulted in reproducible complement activation via the classical pathway of complement and albuminuria in wild-type mice. Because albuminuria was absent in FcR-gamma-chain(-/-) mice and reduced in C3(-/-) mice, a role for both FcgammaR and complement is postulated. Because C1q(-/-) and C4(-/-) mice lacking a functional classical and lectin pathway did develop albuminuria, we suggest involvement of the alternative pathway of complement. Anti-GBM glomerulonephritis occurs acutely following the administration of mouse anti-rabbit IgG, and proceeds in a chronic fashion dependent on both FcgammaR and complement. This novel attenuated model allows elucidating the relative contribution of different mediator systems of the immune system to the development of renal injury, and also provides a platform for the assessment of different treatment protocols and evaluation of drugs that ultimately may be beneficial for the treatment of anti-GBM mediated glomerulonephritides.

Induction of donor-specific T-cell hyporesponsiveness using dexamethasone-treated dendritic cells in two fully mismatched rat kidney transplantation models.

BACKGROUND: Dendritic cells (DC) can exert powerful immune stimulatory as well as regulatory functions and are therefore important tools for therapeutic strategies. Dexamethasone (Dex) was previously shown to inhibit DC maturation and to induce regulatory properties both in vitro and in vivo. Here, we investigated the immunoregulatory role of DexDC in two different rat acute rejection models of kidney transplantation. METHODS: Rat DC were generated from BN and DA bone marrow in the presence of the corticosteroid, Dex. The function of Dex-modulated DC was analyzed in vitro and in vivo, using a BN to LEW and a DA to LEW renal transplantation model in the absence of other forms of immunosuppression. T cells of transplanted rats were isolated and restimulated with donor mature DC (lipopolysaccharide [LPS] or CD40L activated). T-cell responsiveness was analyzed by proliferative capacity and IFN-gamma production. RESULTS: Stimulation of Dex-modulated rat DC with LPS resulted in normal IL-10 production, whereas synthesis of IL-12 was impaired. In accordance, the capacity of LPS-DexDC to stimulate T-cell activation was decreased. In both renal transplantation models, treatment with donor-derived LPS-DexDC induced a significant donor-specific T-cell hyporesponse. However, pretreatment did not result in a prolonged graft survival. CONCLUSIONS: In two fully mismatched kidney transplantation models, donor-derived LPS-DexDC induce a donor-specific T-cell hyporesponse. However, in this setting allograft survival was not improved, suggesting an important role for T cells with indirect alloreactivity. Understanding the underlying mechanism involved in the rejection process will improve the development of a cell-based immunotherapy.

Mannose-binding lectin engagement with late apoptotic and necrotic cells.

The serum opsonin mannose-binding lectin (MBL) has been shown to be involved in the handling of apoptotic cells. However, at what stage in the process this happens and whether this mediates activation of complement is unknown. Cells rendered apoptotic or necrotic were incubated with purified MBL/MBL-associated serine protease (MASP) complexes and assessed by flow cytometry and fluorescence microscopy. MBL bound specifically to late apoptotic cells, as well as to apoptotic blebs and to necrotic cells, but not to early apoptotic cells. Binding of MBL could be inhibited by EDTA as well as with an antibody against the CRD region. Addition of C1q, another serum opsonin involved in the handling of apoptotic cells, prior to MBL partly inhibited MBL binding to apoptotic cells and vice versa. MBL/MASP could initiate deposition of purified complement C4 on the target cells. However, addition of MBL/MASP to whole serum deficient for both C1q and MBL did not enhance deposition of C4, but MBL enhanced phagocytosis of apoptotic cells by macrophages. These results demonstrate that MBL interacts with structures exposed on cells rendered late apoptotic or necrotic and facilitates uptake by macrophages. Thus, MBL may promote non-inflammatory sequestration of dying host cells.