Time course of costamere-related alterations in focal adhesion signaling and composition of rat soleus muscle after achilles tenotomy.

Sarcolemma-based focal adhesions (costameres) are a central hub for the cytoskeletal anchoring of myofibrils and mechano-regulated signaling. Here we report the time course of alterations in focal adhesion-associated signaling and fiber composition in rat soleus muscle after Achilles tenotomy. The report includes data from tenotomized muscles and contralateral mock controls to expose whether muscle degeneration after tenotomy is due to the transection of the Achilles tendon, or circumjacent surgical manipulations of the tendon. With respect to the interpretation of the data regarding mechanistic implications of costamere-associated processes for surgical repair of the detached muscle-tendon complex the reader is referred to the accompanying research article 'Focal adhesion kinase coordinates costamere-related JNK signaling with muscle fiber transformation after Achilles tenotomy and tendon reconstruction' Ferrié et al., 2019.

Focal adhesion kinase coordinates costamere-related JNK signaling with muscle fiber transformation after Achilles tenotomy and tendon reconstruction.

Achilles tendon rupture necessitates rapid tendon reattachment to reinstate plantar flexion before affected muscles deteriorate through muscle fiber atrophy and transformation. The implicated process may involve alterations in sarcolemmal sites of myofibril attachment (costameres), which control myofibrillogenesis via a mechano-regulated mechanism through integrin-associated focal adhesion kinase (FAK). We assessed the contribution of FAK to alterations in fiber type composition and expression of costamere-associated structural proteins, the phosphorylation status of Y397-FAK and downstream mTOR/JNK-P70S6K hypertrophy signaling in rat soleus muscle after Achilles tenotomy and tendon repair. Achilles tenotomy induced a profound deterioration of muscle composition 14 days, but not 4 days, following tendon release, comprising specifically increased area percentages of fast type fibers, fibers with internal nuclei, and connective tissue. Concomitantly, expression of costameric proteins FAK and meta-vinculin, and phosphorylation of T421/S424-P70S6K and T183/Y185-JNK was elevated, all of which was mitigated by tendon reattachment immediately after release. Overexpression of FAK in soleus muscle fibers and reattachment corrected the expression of meta- and gamma-vinculin isoforms to the lower levels in mock controls while further enhancing T183/Y185-JNK phosphorylation and levels of FAK C-terminus-related inhibitory proteins. Co-overexpression of the FAK inhibitor, FRNK, lowered FAK-overexpression driven Y397-FAK phosphorylation and T183/Y185-JNK phosphorylation. FAK levels correlated to molecular and cellular hallmarks of fiber degeneration. The findings demarcate the window between 4 and 14 days after tenotomy as costamere-dependent muscle transformation process, and expose that FAK overexpression prevents molecular aspects of the pathology which within the study limitations does not result in the mitigation of muscle fiber degeneration.250 words.

CaMKII content affects contractile, but not mitochondrial, characteristics in regenerating skeletal muscle.

BACKGROUND: The multi-meric calcium/calmodulin-dependent protein kinase II (CaMKII) is the main CaMK in skeletal muscle and its expression increases with endurance training. CaMK family members are implicated in contraction-induced regulation of calcium handling, fast myosin type IIA expression and mitochondrial biogenesis. The objective of this study was to investigate the role of an increased CaMKII content for the expression of the contractile and mitochondrial phenotype in vivo. Towards this end we attempted to co-express alpha- and beta-CaMKII isoforms in skeletal muscle and characterised the effect on the contractile and mitochondrial phenotype. RESULTS: Fast-twitch muscle m. gastrocnemius (GM) and slow-twitch muscle m. soleus (SOL) of the right leg of 3-month old rats were transfected via electro-transfer of injected expression plasmids for native α/ß CaMKII. Effects were identified from the comparison to control-transfected muscles of the contralateral leg and non-transfected muscles. α/ß CaMKII content in muscle fibres was 4-5-fold increased 7 days after transfection. The transfection rate was more pronounced in SOL than GM muscle (i.e. 12.6 vs. 3.5%). The overexpressed α/ß CaMKII was functional as shown through increased threonine 287 phosphorylation of ß-CaMKII after isometric exercise and down-regulated transcripts COXI, COXIV, SDHB after high-intensity exercise in situ. α/ß CaMKII overexpression under normal cage activity accelerated excitation-contraction coupling and relaxation in SOL muscle in association with increased SERCA2, ANXV and fast myosin type IIA/X content but did not affect mitochondrial protein content. These effects were observed on a background of regenerating muscle fibres. CONCLUSION: Elevated CaMKII content promotes a slow-to-fast type fibre shift in regenerating muscle but is not sufficient to stimulate mitochondrial biogenesis in the absence of an endurance stimulus.

Dynamic patterns of ventricular remodeling and apoptosis in hearts unloaded by heterotopic transplantation.

BACKGROUND: Mechanical unloading of failing hearts can trigger functional recovery but results in progressive atrophy and possibly detrimental adaptation. In an unbiased approach, we examined the dynamic effects of unloading duration on molecular markers indicative of myocardial damage, hypothesizing that potential recovery may be improved by optimized unloading time. METHODS: Heterotopically transplanted normal rat hearts were harvested at 3, 8, 15, 30, and 60 days. Forty-seven genes were analyzed using TaqMan-based microarray, Western blot, and immunohistochemistry. RESULTS: In parallel with marked atrophy (22% to 64% volume loss at 3 respectively 60 days), expression of myosin heavy-chain isoforms (MHC-α/-ß) was characteristically switched in a time-dependent manner. Genes involved in tissue remodeling (FGF-2, CTGF, TGFb, IGF-1) were increasingly upregulated with duration of unloading. A distinct pattern was observed for genes involved in generation of contractile force; an indiscriminate early downregulation was followed by a new steady-state below normal. For pro-apoptotic transcripts bax, bnip-3, and cCasp-6 and -9 mRNA levels demonstrated a slight increase up to 30 days unloading with pronunciation at 60 days. Findings regarding cell death were confirmed on the protein level. Proteasome activity indicated early increase of protein degradation but decreased below baseline in unloaded hearts at 60 days. CONCLUSIONS: We identified incrementally increased apoptosis after myocardial unloading of the normal rat heart, which is exacerbated at late time points (60 days) and inversely related to loss of myocardial mass. Our findings suggest an irreversible detrimental effect of long-term unloading on myocardium that may be precluded by partial reloading and amenable to molecular therapeutic intervention.

Electric pulses augment reporter gene expression in the beating heart.

BACKGROUND: Gene therapy of the heart has been attempted in a number of clinical trials with the injection of naked DNA, although quantitative information on myocellular transfection rates is not available. The present study aimed to quantify the efficacy of electropulsing protocols that differ in pulse duration and number to stimulate transfection of cardiomyocytes and to determine the impact on myocardial integrity. METHODS: Reporter plasmid for constitutive expression of green fluorescent protein (GFP) was injected into the left ventricle of beating hearts of adult, male Lewis rats. Four electrotransfer protocols consisting of repeated long pulses (8 × 20 ms), trains of short pulses (eight trains of either 60 or 80 × 100 µs) or their combination were compared with control procedures concerning the degree of GFP expression and the effect on infiltration, fibrosis and apoptosis. RESULTS: All tested protocols produced GFP expression at the site of plasmid injection. Continuous pulses were most effective and increased the number of GFP-positive cardiomyocytes by more than 300-fold compared to plasmid injection alone (p < 0.05). Concomitantly, the incidence of macrophage infiltration, fibrosis and cell death was increased. Trains of short pulses reduced macrophage infiltration and fibrosis by four- and two-fold, respectively, although they were 20-fold less efficient in stimulating cardiomyocyte transfection. GFP expression co-related to delivered electric energy, infiltration and fibrosis, although not apoptosis. CONCLUSIONS: The data imply that electropulsing of the myocardium promotes the overexpression of exogenous protein in mature cardiomyocytes in relation to an injury component. Fractionation of pulses is indicated as a option for sophisticated gene therapeutic approaches to the heart.