The Ca2+-ATPase of sarco(endo)plasmic reticulum (SERCA) is a protein of about 110 kDa member of the P-type ATPases family. SERCA pumps utilize the energy derived from the hydrolysis of a molecule of ATP to transport two Ca2+ ions across the Sarcoplasmic Reticulum (SR) membrane to decrease the Ca2+ concentration in the cytosol. SERCA isoform 1a (SERCA1a) is the mainly expressed isoform in adult fast-twitch muscle fibre and it is both structurally and functionally the best characterized member of the P-type ion translocating ATPases.
An inherited muscle disorder defined as Bovine Congenital Pseudomyotonia (PMT) has been recently described in two important Italian cattle breeds Chianina and Romagnola (RMG) and, as a single case, in a Dutch improved Red&White (VRB) crossbred calf in the Netherlands. Clinically the disorder is characterized by an exercise-induced muscle contraction not associated to electromyogram alterations. Basing on the clinical signs, the explanation proposed for the delayed relaxation of muscle was a prolonged elevation in Ca2+ ion cytoplasmic concentration. Because the structure primary involved in the re-uptake of the Ca2+ ion into the SR after muscle-contraction is the ATPase pump, the more likely aetiological hypothesis was an involvement of SERCA1a protein. Indeed, DNA sequencing of affected animals provided evidence of mutations in ATP2A1 gene coding for SERCA1. So far, different point mutations have been described in the different breeds that displayed the disease.
Bovine PMT has been well characterized at both genetic and biochemical levels in Chianina breed. The main objective of this project is to complete the characterization of the disease by mean of analysing from a morphological, histochemical and biochemical point of view the other PMT-cases diagnosed in RMG and VRB breeds. The final aims are to increase the knowledge on morphological and biochemical effects of the disease on muscle tissue, to evaluate phenotypic variability of the disease and to identify possible adaptive/compensatory mechanisms that could drive the future research to develop new therapeutic approaches. The interest on bovine PMT arises from its similarities with a rare human inherited muscular dysfunction named Brody disease. Indeed, PMT-affected Chianina cattle has been accepted as an interesting and useful non-conventional animal model for the study of this human pathological condition.
The histopathological examinations on semimembranosus muscle biopsies from RMG and VRB affected animals did not show severe signs of muscle damage. Some pale degenerated fibres, filled or surrounded by macrophages, were identified within the tissue, although they were always accompanied by regenerative phenomena (small size fibres neonatal MHC-positive). Histochemical protocols (COX-SDH and PAS staining) did not reveal metabolic alterations or changes in glycogen distribution.
Immunohistochemical investigations on SERCA1 expression showed that pathological cases had a decrease in immunoreactivity for SERCA1 antibody suggesting a lower presence of the protein. The latter finding was confirmed by biochemical analysis on microsomal fractions enriched in content of SR membranes. Immunoblotting proved that only SERCA1 protein had a reduction in quantity whereas nor junctional (RyR) nor non-junctional (SL and GP53) markers of SR showed any variance. The quantitative decrease was also associated with a lower activity of the protein demonstrated by Ca2+ATPase activity assay. Real Time RT-PCR assays demonstrated that mRNA for SERCA1 was normally expressed suggesting that the protein reduction was due to post-transcriptional events.
Interestingly, the sections of the affected VRB subject showed an increased and more homogeneous stain for COX and SDH suggesting an augmentation in mitochondria content. This finding was reinforced by immunohistochemical and immunoblot surveys using an antibody against a protein of the mitochondrial outer membrane (Tom20). Moreover, microsomal fractions from VRB animal showed an increase in plasma membrane Ca2+ATPase (PMCA) when compare with controls.
Finally in RMG and VRB affected subjects some fibres were immunoreactive for SERCA1 and phospholamban (PLB, regulator protein of SERCA2) or SERCA isoforms 1 and 2 suggesting an alteration in the classic mutually exclusive protein expression pattern (either SERCA1 or SERCA2 and PLB).
The results of this research describe Bovine Congenital Pseudomyotonia in RGM and VRB as a muscular disease characterized by mild histopathological alterations and a sole decrease in SERCA1 content and activity, in full agreement with the studies on Chianina. All the analyses performed on VRB and RGM animals allow to obtain a complete picture of the PMT in these breeds and afford to define a general histopathological and biochemical case of PMT. In addition, for the first time compensatory mechanisms were demonstrated providing histochemical and biochemical evidences to support mitochondria and PMCA involvement in the Ca2+ homeostasis during the pathological condition caused by PMT mutation. Finally, the first evidence of co-expression of SERCA1, SERCA2 and PLB in affected animals, was an intriguing collateral finding that suggests a great potential plasticity of cattle muscle resembling the human muscle once again.
A side project was developed in collaboration with the group of Prof. R. Smith and Dr. J. Dudhia of the Clinical Sciences and Services Department of the Royal Veterinary College (London). Two different pilot studies were carry out on extracellular matrix response to tendon injury in equine. The first had as object of study the cartilage oligomeric matrix protein (COMP) recently proposed as useful biomarker of tendinopathy. The aim of this project was to assess the level of fragmentation (index of alteration but also re-modelling) of COMP in healthy tendon tissue collected in sites well-known to be more frequently involved in lesions (predisposed to injury). The tendons analysed were the superficial digital flexor (SDFT) and the deep digital flexor (DDFT). Immunoblotting and densitometric analysis of total protein extracts showed an higher COMP fragmentation level in healthy predisposed sites when compare with non-predisposed sites. Moreover, for the first time the different patterns of COMP expression in SDFT and DDFT of young and old subjects were described. The second pilot study analysed the morphological aspect of tendon tissue affected by tear-type lesion. The objective was to evaluate the influence of the lesion on the tissue adjacent, far and remote to the injury site. The tendon was extremely altered close to the lesion while the other areas showed high cellularity and vascularity but normal matrix organization. Masson trichrome staining distinguished damaged tissue (blue) from healthy (red) running to become an intriguing tool for histological investigation. Immunohistochemistry showed an increased reactivity for COMP antibody in the areas more proximal to the tear reinforcing the studies that support a fundamental role of this protein in the matrix re-modelling. Increasing the knowledge of COMP distribution in SDFT and DDFT, and providing information of tendon response to tear lesion, these two pilot studies create a useful platform for future researches with possible clinical and therapeutic implication
