Stephanie E. Edelmann

Transformation of adult rat cardiac myocytes in primary culture

We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long‐term cell culture. Morphometric parameters, sarcomere length, T‐tubule density, cell capacitance, L‐type calcium current (ICa,L), inward rectifier potassium current (IK1), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 ± 70 μm2 on day 0 to 1432 ± 50 μm2 on day 5), T‐tubule density, systolic shortening (from 0.11 ± 0.02 to 0.05 ± 0.01 μm) and amplitude of calcium transients (from 1.54 ± 0.19 to 0.67 ± 0.19) over 5 days of culture. The negative force–frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 ± 8 to 105 ± 11 pF) and membrane currents were also reduced (ICa,L, from 3.98 ± 0.39 to 2.12 ± 0.37 pA pF; and IK1, from 34.34p ± 2.31 to 18.00 ± 5.97 pA pF−1). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 ± 2.5 to 34.2 ± 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.

Tricellulin deficiency affects tight junction architecture and cochlear hair cells

The two compositionally distinct extracellular cochlear fluids, endolymph and perilymph, are separated by tight junctions that outline the scala media and reticular lamina. Mutations in TRIC (also known as MARVELD2), which encodes a tricellular tight junction protein known as tricellulin, lead to nonsyndromic hearing loss (DFNB49). We generated a knockin mouse that carries a mutation orthologous to the TRIC coding mutation linked to DFNB49 hearing loss in humans. Tricellulin was absent from the tricellular junctions in the inner ear epithelia of the mutant animals, which developed rapidly progressing hearing loss accompanied by loss of mechanosensory cochlear hair cells, while the endocochlear potential and paracellular permeability of a biotin-based tracer in the stria vascularis were unaltered. Freeze-fracture electron microscopy revealed disruption of the strands of intramembrane particles connecting bicellular and tricellular junctions in the inner ear epithelia of tricellulin-deficient mice. These ultrastructural changes may selectively affect the paracellular permeability of ions or small molecules, resulting in a toxic microenvironment for cochlear hair cells. Consistent with this hypothesis, hair cell loss was rescued in tricellulin-deficient mice when generation of normal endolymph was inhibited by a concomitant deletion of the transcription factor, Pou3f4. Finally, comprehensive phenotypic screening showed a broader pathological phenotype in the mutant mice, which highlights the non-redundant roles played by tricellulin.

Age-Associated Disruption of Molecular Clock Expression in Skeletal Muscle of the Spontaneously Hypertensive Rat

It is well known that spontaneously hypertensive rats (SHR) develop muscle pathologies with hypertension and heart failure, though the mechanism remains poorly understood. Woon et al. (2007) linked the circadian clock gene Bmal1 to hypertension and metabolic dysfunction in the SHR. Building on these findings, we compared the expression pattern of several core-clock genes in the gastrocnemius muscle of aged SHR (80 weeks; overt heart failure) compared to aged-matched control WKY strain. Heart failure was associated with marked effects on the expression of Bmal1, Clock and Rora in addition to several non-circadian genes important in regulating skeletal muscle phenotype including Mck, Ttn and Mef2c. We next performed circadian time-course collections at a young age (8 weeks; pre-hypertensive) and adult age (22 weeks; hypertensive) to determine if clock gene expression was disrupted in gastrocnemius, heart and liver tissues prior to or after the rats became hypertensive. We found that hypertensive/hypertrophic SHR showed a dampening of peak Bmal1 and Rev-erb expression in the liver, and the clock-controlled gene Pgc1α in the gastrocnemius. In addition, the core-clock gene Clock and the muscle-specific, clock-controlled gene Myod1, no longer maintained a circadian pattern of expression in gastrocnemius from the hypertensive SHR. These findings provide a framework to suggest a mechanism whereby chronic heart failure leads to skeletal muscle pathologies; prolonged dysregulation of the molecular clock in skeletal muscle results in altered Clock, Pgc1α and Myod1 expression which in turn leads to the mis-regulation of target genes important for mechanical and metabolic function of skeletal muscle.

Disruption of Circadian Gene Expression in Skeletal Muscle but not Liver in Pre-Hypertensive SHR Vs. WKY Rats

Recently, alterations of the molecular clock and circadian rhythms have been implicated as contributing factors to cardiovascular and skeletal muscle disease. Woon et al. (2007) determined that a polymorphism found in the congenic interval of the SHR rat is associated with hypertension and type II diabetes. Here, we examined the expression of circadian genes in striated muscle (cardiac and skeletal muscle) in young pre-hypertensive SHR (6 weeks old) and age-matched Wistar-Kyoto (WKY) male rats. The rats were entrained to a 12 hour light: 12 hour dark cycle for 2 weeks and then placed in constant darkness for 30 hours. Cardiac muscle (left ventricle), skeletal muscle (soleus) and non-muscle tissue (liver) were collected every 4 hours for 40 hrs, totally 10 time points. Expression of core clock genes (Bmal1, Clock, Per2, Rorα, Rev-erb) and the clock-controlled gene, Dbp, were analyzed using real-time quantitative PCR. Expression of Bmal1 has a clear circadian pattern in muscle and liver tissue of rats. The pattern and amplitude of circadian expression of Bmal1 were not altered between WKY and SHR strains in every tissue studied. In contrast, expression of the other clock genes, Rorα, Dbp, Rev-erb, Clock and Per2, were significantly dys-regulated in the soleus muscle from the SHR rat. In the left ventricle, circadian expression of Per2 was dampened in the SHR but the other clock genes were unchanged. In liver, there were no differences in expression of any of the clock genes between the SHR and WKY rats. These data suggest that components of the molecular clock are disrupted in striated muscle prior to overt signs of hypertension. The contribution of this disruption in the clock to hypertension and type II diabetes are to be determined.