Nonmuscle myosin heavy-chain gene MYH14 is expressed in cochlea and mutated in patients affected by autosomal dominant hearing impairment (DFNA4)

Myosins have been implicated in various motile processes, including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Different members of the myosin superfamily are responsible for syndromic and nonsyndromic hearing impairment in both humans and mice. MYH14 encodes one of the heavy chains of the class II nonmuscle myosins, and it is localized within the autosomal dominant hearing impairment (DFNA4) critical region. After demonstrating that MYH14 is highly expressed in mouse cochlea, we performed a mutational screening in a large series of 300 hearing-impaired patients from Italy, Spain, and Belgium and in a German kindred linked to DFNA4. This study allowed us to identify a nonsense and two missense mutations in large pedigrees, linked to DFNA4, as well as a de novo allele in a sporadic case. Absence of these mutations in healthy individuals was tested in 200 control individuals. These findings clearly demonstrate the role of MYH14 in causing autosomal dominant hearing loss and further confirm the crucial role of the myosin superfamily in auditive functions.     

Quantification of cytoskeletal deformation in living cells based on hierarchical feature vector matching

The cytoskeleton is a dynamicscaffold in living cells even in the absence of externally imposedforces. In this study on cytoskeletal deformation, the applicability ofhierarchical feature vector matching (HFVM), a new matching method,currently applied in space research and three-dimensional surfacereconstruction, was investigated. Stably transfected CHO-K1 cellsexpressing green fluorescent protein (GFP) coupled to vimentin wereused to visualize spontaneous movement of the vimentin cytoskeleton ofindividual cells using a confocal laser scanning system. We showedthat, with proper parameter and configuration settings, HFVM couldrecognize and trace 60-70% of all image points in artificiallytranslated, rotated, or deformed images. If only points belonging tothe cytoskeleton were selected for matching purposes, the percentage ofmatched points increased to 98%. This high percentage of recognitionalso could be reached in a time series of images, in which a certain degree of bleaching of the fluorescence over the recording time of 30 min was inevitable. In these images, HFVM allowed the detection as wellas the quantification of spontaneous cytoskeletal movements of up to10% of the cell width. Therefore, HFVM appears to be a reliable methodof quantifying dynamic cytoskeletal behavior in living cells.

     

Heat stress pretreatment mitigates postischemic arachidonic acid accumulation in rat heart

Heat stress pretreatment of the heart is known to protect this organ against an ischemic/reperfusion insult 24 h later. Degradation of membrane phospholipids resulting in tissue accumulation of polyunsaturated fatty acids, such as arachidonic acid, is thought to play an important role in the multifactorial process of ischemia/reperfusion-induced damage.The present study was conducted to test the hypothesis that heat stress mitigates the postischemic accumulation of arachidonic acid in myocardial tissue, as a sign of enhanced membrane phospholipid degradation. The experiments were performed on hearts isolated from rats either 24 h after total body heat treatment (42°C for 15 min) or 24 h after sham treatment (control). Hearts were made ischemic for 45 min and reperfused for another 45 min.Heat pretreatment resulted in a significant improvement of postischemic hemodynamic performance of the isolated rat hearts. The release of creatine kinase was reduced from 30 ± 14 (control group) to 17 ± 5 units/g wet wt per 45 min (heat-pretreated group) (p < 0.05). Moreover, the tissue content of the inducible heat stress protein HSP70 was found to be increased 3-fold 24 h after heat treatment. Preischemic tissue levels of arachidonic acid did not differ between heat-pretreated and control hearts. The postischemic ventricular content of arachidonic acid was found to be significantly reduced in heat-pretreated hearts compared to sham-treated controls (6.6 ± 3.3. vs. 17.8 ± 12.0 nmol/g wet wt). The findings suggest that mitigation of membrane phospholipid degradation is a potential mechanism of heat stress-mediated protection against the deleterious effects of ischemia and reperfusion on cardiac cells.     

Heat pretreatment differentially affects cardiac fatty acid accumulation during ischemia and postischemic reperfusion

We investigated whether the cardioprotection induced by heat stress (HS) pretreatment is associated with mitigation of phospholipid degradation during the ischemic and/or postischemic period. The hearts, isolated from control rats and from heat-pretreated rats (42 degrees C for 15 min) either 30 min (HS0.5-h) or 24 h (HS24-h) earlier, were subjected to 45 min of no-flow ischemia, followed by 45 min of reperfusion. Unesterified arachidonic acid (AA) accumulation was taken as a measure for phospholipid degradation. Significantly improved postischemic ventricular functional recovery was only found in the HS24-h group. During ischemia, AA accumulated comparably in control and both HS groups. During reperfusion in control and HS0.5-h hearts, AA further accumulated (control hearts from 82 +/- 33 to 109 +/- 51 nmol/g dry wt, not significant; HS-0.5h hearts from 52 +/- 22 to 120 +/- 53 nmol/g dry wt; P < 0.05). In contrast, AA was lower at the end of the reperfusion phase in HS24-h hearts than at the end of the preceding ischemic period (74 +/- 18 vs. 46 +/- 23 nmol/g dry wt; P < 0.05). Thus accelerated reperfusion-induced degradation of phospholipids in control hearts is completely absent in HS24-h hearts. Furthermore, the lack of functional improvement in HS0.5-h hearts is also associated with a lack of beneficial effect on lipid homeostasis. Therefore, it is proposed that enhanced membrane stability during reperfusion is a key mediator in the heat-induced cardioprotection.     

ADP-ribose stimulates the calcium release channel RyR1 in skeletal muscle of rat

The Ca(2+) mobilizing metabolite cyclic ADP-ribose has been shown to release Ca(2+) from intracellular ryanodine sensitive stores in many cells. However, the activation of the ryanodine receptor of skeletal muscle by cADP-ribose (cADPr) and its precursor and metabolite (beta-NAD(+) and ADPr) remains to be discussed. We studied the effect of ADPr on the Ca(2+) release channel of skeletal muscle RyR1 after incorporation of microsomes isolated from fast muscles of rat in planar lipid bilayers. We observed an increase in the electrophysiological activity of the channel after addition of ADPr (10 microM) at micromolar Ca(2+) concentrations, characterized by a time-lag. The increase in P(o) is mainly due to an increase in the open frequency. The long time course observed for the development of the ADPr effect may indicate that this activation induces a change in the conformation of the RyR1 channel, which increases its sensitivity to calcium.     

GJB2 mutations and degree of hearing loss: a multicenter study

Hearing impairment (HI) affects 1 in 650 newborns, which makes it the most common congenital sensory impairment. Despite extraordinary genetic heterogeneity, mutations in one gene, GJB2, which encodes the connexin 26 protein and is involved in inner ear homeostasis, are found in up to 50% of patients with autosomal recessive nonsyndromic hearing loss. Because of the high frequency of GJB2 mutations, mutation analysis of this gene is widely available as a diagnostic test. In this study, we assessed the association between genotype and degree of hearing loss in persons with HI and biallelic GJB2 mutations. We performed cross-sectional analyses of GJB2 genotype and audiometric data from 1,531 persons, from 16 different countries, with autosomal recessive, mild-to-profound nonsyndromic HI. The median age of all participants was 8 years; 90% of persons were within the age range of 0-26 years. Of the 83 different mutations identified, 47 were classified as nontruncating, and 36 as truncating. A total of 153 different genotypes were found, of which 56 were homozygous truncating (T/T), 30 were homozygous nontruncating (NT/NT), and 67 were compound heterozygous truncating/nontruncating (T/NT). The degree of HI associated with biallelic truncating mutations was significantly more severe than the HI associated with biallelic nontruncating mutations (P<.0001). The HI of 48 different genotypes was less severe than that of 35delG homozygotes. Several common mutations (M34T, V37I, and L90P) were associated with mild-to-moderate HI (median 25-40 dB). Two genotypes--35delG/R143W (median 105 dB) and 35delG/dela(GJB6-D13S1830) (median 108 dB)--had significantly more-severe HI than that of 35delG homozygotes.     

A new autosomal recessive form of Stickler syndrome is caused by a mutation in the COL9A1 gene

Stickler syndrome is characterized by ophthalmic, articular, orofacial, and auditory manifestations. It has an autosomal dominant inheritance pattern and is caused by mutations in COL2A1, COL11A1, and COL11A2. We describe a family of Moroccan origin that consists of four children with Stickler syndrome, six unaffected children, and two unaffected parents who are distant relatives (fifth degree). All family members were clinically investigated for ear, nose, and throat; ophthalmologic; and radiological abnormalities. Four children showed symptoms characteristic of Stickler syndrome, including moderate-to-severe sensorineural hearing loss, moderate-to-high myopia with vitreoretinopathy, and epiphyseal dysplasia. We considered the COL9A1 gene, located on chromosome 6q13, to be a candidate gene on the basis of the structural association with collagen types II and XI and because of the high expression in the human inner ear indicated by cDNA microarray. Mutation analysis of the coding region of the COL9A1 gene showed a homozygous R295X mutation in the four affected children. The parents and four unaffected children were heterozygous carriers of the R295X mutation. Two unaffected children were homozygous for the wild-type allele. None of the family members except the homozygous R295X carriers had any signs of Stickler syndrome. Therefore, COL9A1 is the fourth identified gene that can cause Stickler syndrome. In contrast to the three previously reported Stickler syndrome-causing genes, this gene causes a form of Stickler syndrome with an autosomal recessive inheritance pattern. This finding will have a major impact on the genetic counseling of patients with Stickler syndrome and on the understanding of the pathophysiology of collagens. Mutation analysis of this gene is recommended in patients with Stickler syndrome with possible autosomal recessive inheritance.     

Adaptation of cardiac structure by mechanical feedback in the environment of the cell: a model study.

In the cardiac left ventricle during systole mechanical load of the myocardial fibers is distributed uniformly. A mechanism is proposed by which control of mechanical load is distributed over many individual control units acting in the environment of the cell. The mechanics of the equatorial region of the left ventricle was modeled by a thick-walled cylinder composed of 6-1500 shells of myocardial fiber material. In each shell a separate control unit was simulated. The direction of the cells was varied so that systolic fiber shortening approached a given optimum of 15%. End-diastolic sarcomere length was maintained at 2.1 microns. Regional early-systolic stretch and global contractility stimulated growth of cellular mass. If systolic shortening was more than normal the passive extracellular matrix stretched. The design of the load-controlling mechanism was derived from biological experiments showing that cellular processes are sensitive to mechanical deformation. After simulating a few hundred adaptation cycles, the macroscopic anatomical arrangement of helical pathways of the myocardial fibers formed automatically. If pump load of the ventricle was changed, wall thickness and cavity volume adapted physiologically. We propose that the cardiac anatomy may be defined and maintained by a multitude of control units for mechanical load, each acting in the cellular environment. Interestingly, feedback through fiber stress is not a compelling condition for such control.