Telethonin deficiency is associated with maladaptation to biomechanical stress in the mammalian heart.

RATIONALE: Telethonin (also known as titin-cap or t-cap) is a 19-kDa Z-disk protein with a unique ß-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonin's in vivo function. OBJECTIVE: Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation. METHODS AND RESULTS: By using a variety of different genetically altered animal models and biophysical experiments we show that contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin-titin cross-links via α-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the proapoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis ("mechanoptosis"). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect that may contribute to enhanced rates of apoptosis found in these hearts. CONCLUSIONS: Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect that may also play a role in human heart failure.

MLP (muscle LIM protein) as a stress sensor in the heart.

Muscle LIM protein (MLP, also known as cysteine rich protein 3 (CSRP3, CRP3)) is a muscle-specific-expressed LIM-only protein. It consists of 194 amino-acids and has been described initially as a factor involved in myogenesis (Arber et al. Cell 79:221-231, 1994). MLP soon became an important model for experimental cardiology when it was first demonstrated that MLP deficiency leads to myocardial hypertrophy followed by a dilated cardiomyopathy and heart failure phenotype (Arber et al. Cell 88:393-403, 1997). At this time, this was the first genetically altered animal model to develop this devastating disease. Interestingly, MLP was also found to be down-regulated in humans with heart failure (Zolk et al. Circulation 101:2674-2677, 2000) and MLP mutations are able to cause hypertrophic and dilated forms of cardiomyopathy in humans (Bos et al. Mol Genet Metab 88:78-85, 2006; Geier et al. Circulation 107:1390-1395, 2003; Hershberger et al. Clin Transl Sci 1:21-26, 2008; Knöll et al. Cell 111:943-955, 2002; Knöll et al. Circ Res 106:695-704, 2010; Mohapatra et al. Mol Genet Metab 80:207-215, 2003). Although considerable efforts have been undertaken to unravel the underlying molecular mechanisms-how MLP mutations, either in model organisms or in the human setting cause these diseases are still unclear. In contrast, only precise knowledge of the underlying molecular mechanisms will allow the development of novel and innovative therapeutic strategies to combat this otherwise lethal condition. The focus of this review will be on the function of MLP in cardiac mechanosensation and we shall point to possible future directions in MLP research.

Collagen synthesis by mesenchymal stem cells and aortic valve interstitial cells in response to mechanical stretch.

OBJECTIVE: The synthesis of appropriate extracellular matrix by cells in tissue engineered heart valve constructs will be important for the maintenance of valve cusp integrity and function. We have examined and compared the capacity of mesenchymal stem cells to synthesise collagen in response to stretch in comparison with native aortic valve interstitial cells. METHODS: Cells were stretched on a Flexercell FX4000 apparatus and total collagen synthesis was measured by the incorporation of [3H]-proline. The effect of stretch on gene expression of different collagen types was assessed by RT-PCR. RESULTS: There was a significant (p<0.01) increase in [3H]-proline incorporation into stretched valve cells at 10%, 14% and 20% stretch. The response of mesenchymal stem cells at 14% stretch was similar to that seen in the valve cells. Incorporation of [3H]-proline into soluble proteins in the cell media was significantly higher (p<0.01) only at 14% and 20% stretch in valve interstitial cells. These effects were shared with mesenchymal stem cells at 14% stretch. RT-PCR experiments demonstrated that 14% stretch up-regulated levels of mRNA for COL3A1 gene (type III collagen) but did not increase the expression of COL1A1 gene (type I collagen) in valve interstitial cells. However, both collagen genes could be detected in non-stretched and stretched mesenchymal stem cells. There was no evidence that the mesenchymal stem cells had started to adopt an osteoblastic cell phenotype in response to stretch. CONCLUSIONS: Collagen synthesis by valve interstitial cells is dependent upon the degree and duration of stretch. This response can be mimicked closely by exposure of mesenchymal stem cells to the same stretching profile. These properties could have important implications for the choice of cells and programme of conditioning with which to tissue engineer heart valves.

Effect of different Ti-6Al-4V surface treatments on osteoblasts behaviour.

The purpose of the present work was to examine the effect of different Ti-6Al-4V surface treatments on osteoblasts behaviour. Previous work in this laboratory has demonstrated that an ageing treatment reduces metal ion release from this alloy compared to standard passivation procedures. In this study. human osteosarcoma MG-63 were used in short-term in vitro tests to assay for cell viability and cell proliferation at 12, 24 and 72 h while SaOS-2 were used in long-term in vitro tests to assay for osteonectin, osteopontin, osteocalcin gene expression, total protein amount (TP). alkaline phosphatase activity (ALP) and fibronectin production (FN) for 1-4 weeks. Epifluorescence microscopy was used to observe SaOS-2 cell morphology. After 24h, there was no difference in MG-63 cell viability proliferation or in SaOS-2 cell morphology between the different surface treatments. For the long-term tests, the aged Ti-6Al4V induced significantly higher cell proliferation than the control Ti-6Al-4V at 72h. At week 1, no difference in the osteonectin, osteopontin, and osteocalcin gene expression was found between samples. The peak of ALP activity appeared earlier at week 2 for the control surface compared with the passivated and aged surfaces. The early increase in ALP activity for the control sample could be a compensatory effect of decreased osteoblasts proliferation. There was no difference in the expression of FN for the different surface treatments. Our present results showed that the different surface treatments, which induced different metal ion release kinetics and surface properties, influenced the cell proliferation and ALP activity of osteoblast cells. Aluminium ions release kinetics as well as presence of vanadium ions may play a major role in influencing the osteoblasts behaviour in the present study.

Large-scale gene expression analysis of osteoblasts cultured on three different Ti-6Al-4V surface treatments.

To improve implant biocompatibility, we developed a simple cost-effective thermal surface treatment allowing an increase in the oxide layer thickness of a titanium (Ti) alloy used in orthopaedic implants. The goal of this study was to test in vitro the reaction of osteoblasts to the developed surface treatment and to compare it to the osteoblast reaction to two other surface treatments currently used in the practice of implant surgery. Quantification of osteoblast gene expression on a large scale was used in this study. The kinetics of gene expression over 120 h was followed for 58 genes to quantify the effect of the developed surface treatment. Twenty eight genes were further selected to compare the effects of surface treatments on osteoblasts. Based on the genes studied, we could propose a general pathway for the cell reaction according to the surface treatments used: (1) metal ion release changes the time course of gene expression in the FAK pathway; (2) once the accumulation of metal ions released from the Ti surface exceeds a threshold value, cell growth is diminished and apoptosis may be activated; (3) PTK up-regulation is also induced by metal ion release; (4) the expression of Bcl-2 family and Bax may suggest that metal ions induce apoptosis. The developed treatment seems to increase the Ti-6Al-4V biocompatibility as highlighted by the lower impact of this treatment by the different pathways studied, on the lower inflammatory reaction that could be induced, as well as by the lower induced osteoblast apoptosis compared to the two other surface treatments.

Regulation of the Nrf2 antioxidant pathway by microRNAs: New players in micromanaging redox homeostasis.

MicroRNAs are now thought to play a central role in the regulation of many diverse aspects of cell biology; however, it remains to be fully elucidated how microRNAs can orchestrate cellular redox homeostasis, which plays a central role in a multitude of physiological and pathophysiological processes. The redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) serves as a "master regulator" of cell survival through the coordinated induction of phase II and antioxidant defense enzymes to counteract oxidative stress and modulate redox signaling events. MicroRNAs are able to "fine-tune" the regulation of processes including those directly interacting with the Nrf2 pathway and the generation of reactive oxygen species (ROS). This review highlights that cellular redox homeostasis can be regulated by microRNAs through their modulation of Nrf2-driven antioxidant gene expression as well as key enzymes that generate ROS, which in turn can alter the biogenesis and processing of microRNAs. Therefore redox sensitive microRNAs or "redoximiRs" add an important regulatory mechanism for redox signaling beyond the well-characterized actions of Nrf2. The potential exists for microRNA-based therapies where diminished antioxidant defenses and dysregulated redox signaling can lead to cardiovascular diseases, cancers, neurodegeneration, and accelerated aging.

Genetics of mechanosensation in the heart.

Mechanosensation (the ultimate conversion of a mechanical stimulus into a biochemical signal) as well as mechanotransduction (transmission of mechanically induced signals) belong to the most fundamental processes in biology. These effects, because of their dynamic nature, are particularly important for the cardiovascular system. Therefore, it is not surprising that defects in cardiac mechanosensation, are associated with various types of cardiomyopathy and heart failure. However, our current knowledge regarding the genetic basis of impaired mechanosensation in the cardiovascular system is beginning to shed light on this subject and is at the centre of this brief review.

Inducible overexpression of sFlt-1 in podocytes ameliorates glomerulopathy in diabetic mice.

OBJECTIVE: Podocyte-specific, doxycycline (DOX)-inducible overexpression of soluble vascular endothelial growth factor (VEGF) receptor-1 (sFlt-1) in adult mice was used to investigate the role of the VEGF-A/VEGF receptor (VEGFR) system in diabetic glomerulopathy. RESEARCH DESIGN AND METHODS: We studied nondiabetic and diabetic transgenic mice and wild-type controls treated with vehicle (VEH) or DOX for 10 weeks. Glycemia was measured by a glucose-oxidase method and blood pressure by a noninvasive technique. sFlt-1, VEGF-A, VEGFR2, and nephrin protein expression in renal cortex were determined by Western immunoblotting; urine sFlt-1, urine free VEGF-A, and albuminuria by enzyme-linked immunosorbent assay; glomerular ultrastructure by electron microscopy; and VEGFR1 and VEGFR2 cellular localization with Immunogold techniques. RESULTS: Nondiabetic DOX-treated transgenic mice showed a twofold increase in cortex sFlt-1 expression and a fourfold increase in sFlt-1 urine excretion (P < 0.001). Urine free VEGF-A was decreased by 50%, and cortex VEGF-A expression was upregulated by 30% (P < 0.04). VEGFR2 expression was unchanged, whereas its activation was reduced in DOX-treated transgenic mice (P < 0.02). Albuminuria and glomerular morphology were similar among groups. DOX-treated transgenic diabetic mice showed a 60% increase in 24-h urine sFlt-1 excretion and an approximately 70% decrease in urine free VEGF-A compared with VEH-treated diabetic mice (P < 0.04) and had lower urine albumin excretion at 10 weeks than VEH-treated diabetic (d) mice: d-VEH vs. d-DOX, geometric mean (95% CI), 117.5 (69-199) vs. 43 (26.8-69) mug/24 h (P = 0.003). Diabetes-induced mesangial expansion, glomerular basement membrane thickening, podocyte foot-process fusion, and transforming growth factor-beta1 expression were ameliorated in DOX-treated diabetic animals (P < 0.05). Diabetes-induced VEGF-A and nephrin expression were not affected in DOX-treated mice. CONCLUSIONS: Podocyte-specific sFlt-1 overexpression ameliorates diabetic glomerular injury, implicating VEGF-A in the pathogenesis of this complication.

Podocyte-specific expression of angiopoietin-2 causes proteinuria and apoptosis of glomerular endothelia.

Angiopoietin-2 (Ang-2) modulates embryonic vascular differentiation primarily by inhibiting the antiapoptotic effects of Ang-1 on endothelia that express the Tie-2 receptor. Ang-2 is transiently expressed by developing glomeruli but is downregulated with normal maturation. Glomerular Ang-2 expression is, however, markedly upregulated in animal models of diabetic nephropathy and glomerulonephritis, both leading causes of human chronic renal disease, affecting 10% of the world population. It was hypothesized that Ang-2 might have significant roles in the pathobiology of glomerular disease. Mice with inducible podocyte-specific Ang-2 overexpression were generated. When the transgene was induced in adults for up to 10 wk, mice had significant increases in both albuminuria and glomerular endothelial apoptosis, with significant decreases of both vascular endothelial growth factor-A and nephrin proteins, critical for maintenance of glomerular endothelia and filtration barrier functional integrity, respectively. There was, however, no significant change of systemic BP, creatinine clearance, or markers of renal fibrosis, and podocytes appeared structurally intact. In kidneys of young animals in which Ang-2 had been upregulated during organogenesis, increased apoptosis occurred in just-formed glomeruli. In vitro, short-term exposure of isolated wild-type murine glomeruli to exogenous Ang-2 led to decreased levels of vascular endothelial growth factor-A protein. These novel results provide insight into molecular mechanisms underlying proteinuric disorders, highlight potentially complex interactions between subsets of glomerular cells, and emphasize how a vascular growth factor that has critical roles in normal development may be harmful when re-expressed in the context of adult disease.