Jonathan C. Jarvis

Micro-computed tomography with iodine staining resolves the arrangement of muscle fibres

We illustrate here microCT images in which contrast between muscle and connective tissue has been achieved by means of staining with iodine. Enhancement is shown to be dependent on the concentration of iodine solution (I(2)KI), time in solution and specimen size. Histological examination confirms that the arrangement of individual muscle fibres can be visualised on the enhanced microCT images, and that the iodine accumulates in the muscle fibres in preference to the surrounding connective tissues. We explore the application of this technique to describe the fibrous structure of skeletal muscle, and conclude that it has the potential to become a non-destructive and cost-effective method for investigating muscle fascicle architecture, particularly in comparative morphological studies.

Cardiomyoplasty. A critical review of experimental and clinical results.

Skeletal muscle cardiac assist (SMCA) is emerging as a promising form of surgical treatment for end-stage heart failure. In one form, cardiomyoplasty, the muscle is wrapped around the heart and then activated by electrical stimulation to augment myocardial contraction.1 Cardiomyoplasty has been performed in more than 400 cases worldwide with variable degrees of success. Despite excellent symptomatic improvement in the majority of patients surviving the procedure, objective hemodynamic effects have not been consistently demonstrated.2,3 The hemodynamic effect of cardiomyoplasty has been the subject of a great deal of experimental and clinical research over the past decade. This article discusses in detail the published results of experimental and clinical cardiomyoplasty, with particular emphasis on hemodynamic effects and limitations of the procedure. Other forms of experimental SMCA, including aortomyoplasty, skeletal muscle ventricles, and biomechanical assist devices, were reviewed recently4-6 and therefore are not discussed here.

Cardiac assistance from skeletal muscle : a critical appraisal of the various approaches

We review here various ways in which cardiac assistance might be derived from a patients own skeletal muscle. Calculations based on experimental data and optimistic estimates of the efficiency of the energy conversions involved suggest that the continuous assist available would be limited to about 2 litres a minute if a muscle were used to energise an electromechanical device. It would be more efficient to couple the energy mechanically or hydraulically, but these approaches still pose problems of anatomical placement, muscle attachment, fluid leakage, and cost. Unless these issues can be addressed, the use of skeletal muscle as an internal power source for mechanical circulatory assist devices will remain an unworkable concept. Configurations that couple skeletal muscle contraction directly to the circulation would be more efficient and less costly. In terms of the energy available, a skeletal muscle ventricle could be designed to provide a continuous partial assist of 1–21/min, with flows of up to 8 1/min sustainable for limited periods. Such an approach offers new possibilities for the surgical treatment of chronic cardiac failure.

Application of Micro-Computed Tomography With Iodine Staining to Cardiac Imaging, Segmentation, and Computational Model Development

Micro-computed tomography (micro-CT) has been widely used to generate high-resolution 3-D tissue images from small animals nondestructively, especially for mineralized skeletal tissues. However, its application to the analysis of soft cardiovascular tissues has been limited by poor inter-tissue contrast. Recent ex vivo studies have shown that contrast between muscular and connective tissue in micro-CT images can be enhanced by staining with iodine. In the present study, we apply this novel technique for imaging of cardiovascular structures in canine hearts. We optimize the method to obtain high-resolution X-ray micro-CT images of the canine atria and its distinctive regions-including the Bachmanns bundle, atrioventricular node, pulmonary arteries and veins-with clear inter-tissue contrast. The imaging results are used to reconstruct and segment the detailed 3-D geometry of the atria. Structure tensor analysis shows that the arrangement of atrial fibers can also be characterized using the enhanced micro-CT images, as iodine preferentially accumulates within the muscular fibers rather than in connective tissues. This novel technique can be particularly useful in nondestructive imaging of 3-D cardiac architectures from large animals and humans, due to the combination of relatively high speed ( ~ 1 h/per scan of the large canine heart) and high voxel resolution (36 μm) provided. In summary, contrast micro-CT facilitates fast and nondestructive imaging and segmenting of detailed 3-D cardiovascular geometries, as well as measuring fiber orientation, which are crucial in constructing biophysically detailed computational cardiac models.

Contrast enhanced micro-computed tomography resolves the 3-dimensional morphology of the cardiac conduction system in mammalian hearts.

The general anatomy of the cardiac conduction system (CCS) has been known for 100 years, but its complex and irregular three-dimensional (3D) geometry is not so well understood. This is largely because the conducting tissue is not distinct from the surrounding tissue by dissection. The best descriptions of its anatomy come from studies based on serial sectioning of samples taken from the appropriate areas of the heart. Low X-ray attenuation has formerly ruled out micro-computed tomography (micro-CT) as a modality to resolve internal structures of soft tissue, but incorporation of iodine, which has a high molecular weight, into those tissues enhances the differential attenuation of X-rays and allows visualisation of fine detail in embryos and skeletal muscle. Here, with the use of a iodine based contrast agent (I2KI), we present contrast enhanced micro-CT images of cardiac tissue from rat and rabbit in which the three major subdivisions of the CCS can be differentiated from the surrounding contractile myocardium and visualised in 3D. Structures identified include the sinoatrial node (SAN) and the atrioventricular conduction axis: the penetrating bundle, His bundle, the bundle branches and the Purkinje network. Although the current findings are consistent with existing anatomical representations, the representations shown here offer superior resolution and are the first 3D representations of the CCS within a single intact mammalian heart.

Fast-to-slow transformation in stimulated rat muscle.

Several previous studies have failed to demonstrate changes due to chronic stimulation in contractile speed of innervated fast rat muscles, and it has been suggested that the adaptive capacity of skeletal muscle in this species is limited. We have reassessed this contention. Fast muscles of the rat hind limb were stimulated continuously at 10 or 20 Hz for 55–61 days. The maximum shortening velocity of the extensor digitorum longus muscles was reduced to 50% of the control value. The proportion of type 1 fibers increased from 4% in control muscle to 34% in stimulated muscles and there was a corresponding reduction in type 2B/D fibers. The proportion of type 2A fibers after stimulation was similar to that in control muscles. These results, taken together with our published analyses of myosin isoform composition of these muscles, show that the mechanisms that control gene expression in response to activity are not exclusive to larger mammals.

Power production and working capacity of rabbit tibialis anterior muscles after chronic electrical stimulation at 10 Hz.

1. The muscles of the distal anterior compartment of the left hindlimb of rabbits were subjected to continuous indirect electrical stimulation at 10 Hz for periods of up to 12 weeks by means of an implantable stimulator. 2. The maximum shortening velocity (Vmax) and the velocity for maximum power production in single contractions (Vopt) were reduced to 42% and 32% of control values respectively after 12 weeks of stimulation. The rate of change of these parameters was greatest between the second and sixth week of stimulation. These changes, it is suggested, reflect the documented time course of the replacement of fast with slow isoforms of myosin. 3. The reductions in force production and speed of the stimulated muscles combined to produce a marked, progressive decline in the maximum power produced in single contractions. After 8 weeks of stimulation, the maximum power output had fallen to less than 10% of the control value. 4. The fatigue resistance of the stimulated and control muscles was tested over several hours of cyclical shortening contractions designed to elicit an initial power output of 10 W kg‐1 with the muscles set to contract at Vopt. This level of work output represented about 1.6% (control) and 25% (12‐week‐stimulated) of the absolute maximum power output achieved during single contractions. 5. Despite the large reduction in the maximum power output of single contractions, the stimulated muscles showed less than 10% reduction in their power output during the fatigue tests over periods of up to 7 h. The control muscles showed a 70% reduction over the same period. There was no difference in the fatigue resistance under this protocol between muscles stimulated for 2 weeks and those stimulated for longer periods. Transformation of myosin isoforms, which is known to occur later than 2 weeks after the start of stimulation, is not necessary for the induction of this degree of fatigue resistance.

Recent advances in management of alkaptonuria (invited review; best practice article)

Alkaptonuria (AKU) is an autosomal recessive condition arising as a result of a genetic deficiency of the enzyme homogentisate 1,2 dioxygenase and characterised by accumulation of homogentisic acid (HGA). Oxidative conversion of HGA leads to production of a melanin-like polymer in a process termed ochronosis. The binding of ochronotic pigment to the connective tissues of the body leads to multisystem disorder dominated by premature severe spondylo-arthropathy. Other systemic features include stones (renal, prostatic, salivary, gall bladder), renal damage/failure, osteopenia/fractures, ruptures of tendons/muscle/ligaments, respiratory compromise, hearing loss and aortic valve disease. Detection of these features requires systematic investigation. Treatment in AKU patients is palliative and unsatisfactory. Ascorbic acid, low protein diet and physiotherapy have been tried but do not alter the underlying metabolic defect. Regular surveillance to detect and treat complications early is important. Palliative pain management is a crucial issue in AKU. Timely spinal surgery and arthroplasty are the major treatment approaches at present. A potential disease modifying drug, nitisinone, inhibits 4-hydroxy-phenyl-pyruvate-dioxygenase and decreases formation of HGA and could prevent or slow the progression of disease in AKU. If nitisinone therapy is able to complement the biochemical ‘cure’ with improved outcomes, it will completely alter the way we approach the management of this disease. Greater efforts to improve recognition and registration of the disease will be worthwhile. Improved laboratory diagnostics to monitor the tyrosine metabolic pathway that includes plasma metabolites including tyrosine to monitor efficacy, toxicity and safety postnitisinone will also be required.

Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1): an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study to investigate the effect of once daily nitisinone on 24-h urinary homogentisic acid excretion in patients with alkaptonuria after 4 weeks of treatment

Background Alkaptonuria (AKU) is a serious genetic disease characterised by premature spondyloarthropathy. Homogentisate-lowering therapy is being investigated for AKU. Nitisinone decreases homogentisic acid (HGA) in AKU but the dose-response relationship has not been previously studied. Methods Suitability Of Nitisinone In Alkaptonuria 1 (SONIA 1) was an international, multicentre, randomised, open-label, no-treatment controlled, parallel-group, dose-response study. The primary objective was to investigate the effect of different doses of nitisinone once daily on 24-h urinary HGA excretion (u-HGA24) in patients with AKU after 4 weeks of treatment. Forty patients were randomised into five groups of eight patients each, with groups receiving no treatment or 1 mg, 2 mg, 4 mg and 8 mg of nitisinone. Findings A clear dose-response relationship was observed between nitisinone and the urinary excretion of HGA. At 4 weeks, the adjusted geometric mean u-HGA24 was 31.53 mmol, 3.26 mmol, 1.44 mmol, 0.57 mmol and 0.15 mmol for the no treatment or 1 mg, 2 mg, 4 mg and 8 mg doses, respectively. For the most efficacious dose, 8 mg daily, this corresponds to a mean reduction of u-HGA24 of 98.8% compared with baseline. An increase in tyrosine levels was seen at all doses but the dose-response relationship was less clear than the effect on HGA. Despite tyrosinaemia, there were no safety concerns and no serious adverse events were reported over the 4 weeks of nitisinone therapy. Conclusions In this study in patients with AKU, nitisinone therapy decreased urinary HGA excretion to low levels in a dose-dependent manner and was well tolerated within the studied dose range. Trial registration number EudraCT number: 2012-005340-24. Registered at ClinicalTrials.gov: NCTO1828463.

The dose-related response of rabbit fast muscle to long-term low-frequency stimulation

Rabbit tibialis anterior muscles were stimulated continuously at 2.5 Hz, 5 Hz, or 10 Hz for 10 months. The resulting adaptive transformation was dose‐related for contractile speed, myosin isoform composition, and enzyme activities. The “fast‐oxidative” state produced by stimulation at 2.5 Hz was stable: even after 10 months, 84% of the fibers were of type 2A. Absence of a secondary decline in oxidative activity in these muscles provided strong evidence of a causal link between myosin transitions and metabolic adaptation. Significant fiber loss occurred only after prolonged stimulation at 10 Hz. The myosin isoform composition of individual muscles stimulated at 5 Hz resembled that of muscles stimulated at either the lower or the higher frequency, behavior consistent with a threshold for fiber type change. In clinical applications such as cardiomyoplasty, muscles could be used more effectively by engineering their properties to combine speed and power of contraction with the necessary resistance to fatigue.