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CONTRACTILE FUNCTIONS OF SLOW AND FAST SKELETAL MUSCLES IN STREPTOZOTOCIN INDUCED TYPE 1 DIABETIC SPRAGUE DAWLEY RATS.

Byline: Kamil Asghar Imam, Muhamamd Mazhar Hussain and Shoaib Bin Aleem

Abstract

Objective: To evaluate the contractile functions of slow and fast skeletal muscles in streptozotocin induced type 1 diabetic male Sprague Dawley rats.

Study Design: Randomized control trial.

Place and Duration: Department of Physiology, Army Medical College, Rawalpindi, from April 2010 to April 2011.

Material and Methods: Thirty healthy male Sprague Dawley rats were divided into two groups. The rats in group I (male control; n = 15) were fed on normal pellet diet and water ad libitum and received single intraperitoneal injection of normal saline at the start of study (day 1). The rats in group II (male diabetic; n = 15) were fed on normal pellet diet and water ad libitum and rendered diabetic by single intraperitoneal injection of streptozotocin (STZ) 65 mg/kg body weight at the start of study (day 1). Development of diabetes was confirmed within 72 hours by measuring blood glucose levels by glucometer. At the end of four weeks, i.e on day 29, dissection of slow soleus and fast extensor digitorum longus (EDL) muscles was carried out. These muscles were selected because they represent two distinctly different fiber type populations, that is, soleus (80% type I, 20% type IIA, 0% type IIB) and EDL (0% type I, 11% type IIA, 89% type IIB).

Their contractile parameters were recorded by iWorx advanced animal/human physiology data acquisition unit (AHK/214), including maximum isometric twitch tension, time to peak twitch tension, time taken to relax to 50% of the peak twitch tension, maximum fused tetanic tension, maximum fused tetanic tension after the fatigue protocol and tetanic tension after 5 minutes of rest period following the fatigue protocol.

Results: After four weeks, no significant difference was found when maximum isometric twitch tension (ITT) in isolated soleus and EDL muscles of the male diabetic group was compared with the control group. Time to peak twitch tension (TPT) and time taken to relax to 50% of the peak twitch tension (HRT) in isolated soleus muscle of the male diabetic group were significantly longer (p<0.001) as compared to the control group. On the contrary, TPT and HRT in isolated EDL muscle of the diabetic group were similar to the control group. Maximum fused tetanic tension in isolated soleus muscle of the diabetic group was similar to the control group. On the contrary, maximum fused tetanic tension in isolated EDL muscle of the male diabetic group was significantly lower (p<0.001) as compared to the control group.

Maximum fused tetanic tension after the fatigue protocol and tetanic tension after 5 minutes of rest period following the fatigue protocol in isolated soleus and EDL muscles of the male diabetic group were significantly lower (p 200 mg/dl)8. Blood glucose was measured at regular intervals after every week, throughout the study, until the completion of study 4 weeks later (day 29). The rats in group I and group II were fed on normal pellet diet and water ad libitum.

At the end of four weeks, the rats were anaesthetized by administering single intraperitoneal injection of sodium pentobarbitone (50 mg/100 g body weight)9. The soleus and extensor digitorum longus (EDL) muscles were dissected free from the surrounding connective tissue10,11. For the measurement of contractile functions, the muscles were mounted in an organ bath containing Krebs-Ringer solution, gassed with 95% O2 - 5% CO2 at 30degC. The proximal tendons of isolated soleus and EDL muscles were alternatively tied to the force transducer (FT-100) connected to iWorx advanced animal/human physiology data acquisition unit (AHK/214). Contractions were evoked by stimulation via platinum electrodes placed directly on to the muscle. Labscribe software was used to collect, digitize, analyze, and store the data to a personal computer. The length of each muscle was adjusted for maximal twitch tension. Passive and twitch tensions were then recorded.

The speed related contractile properties were monitored by measuring time to peak twitch tension and time taken to relax to 50% of the peak twitch tension. The force-frequency relationship was determined by using stimulations of 1 second. Stimulation frequencies of 5-90 Hz for the isolated soleus muscle and 5-110 Hz for the isolated EDL muscle were used. Rest period of 3 minutes was allowed between each stimulus. The maximum fused tetanic tension was then recorded. The fatigue characteristics of each muscle were determined by stimulating the muscle with optimum frequency for 1 second with 5 seconds rest period in between, for the total period of 5 minutes. A measure of recovery from fatigue was also made by recording the tetanic tension after the 5 minutes rest period following the fatigue protocol12. All measured forces were expressed as Newton per gram (N/g) wet muscle mass13.

Data Analysis

Data was entered into SPSS version 18. Mean and standard deviation (SD) were calculated for skeletal muscle function variables. The statistical significance of difference between the groups was determined by applying independent sample's t-test. The difference was considered significant if p-value was found less than 0.05.

RESULTS

At the end of four weeks of study, that is, on day 29, blood glucose level in the male diabetic group (302.67 +- 4.89 mg/dl) was significantly higher (p < 0.001) as compared to the male control group (113.47 +- 4.07 mg/dl). The contractile properties of isolated soleus muscle in the male diabetic rats and healthy controls have been compared in table 1.

No significant difference was found between cases and controls in maximum isometric twitch tension (p=0.153) and maximum fused tetanic tension (p=0.126). Time to peak twitch tension (TPT) and time taken to relax to 50% of the peak twitch tension (HRT) in isolated soleus muscle of the male diabetic group was significantly longer (p<0.001) as compared to the male control group. Maximum fused tetanic tension after the fatigue protocol in isolated soleus muscle of the male diabetic group was significantly less (p < 0.001) as compared to the male control group. Similarly, tetanic tension after 5 minutes of rest period following the fatigue protocol in isolated soleus muscle of the male diabetic group was significantly lower (p<0.001) as compared to the male control group.

The contractile properties of isolated extensor digitorum longus (EDL) muscle in male diabetic rats and healthy controls have been compared in table 2.

Isometric twitch tension (ITT) in isolated extensor digitorum longus (EDL) muscle of the male diabetic and control groups was similar (p=0.062) with no significant difference. Time to peak twitch tension (TPT) in isolated EDL muscle of the male diabetic group was similar (p=0.342) to the male control group. Similarly, time taken to relax to 50% of the peak twitch tension (HRT) in isolated EDL muscle of the male diabetic group was similar (p=0.677) to the male control group with no significant difference. Maximum fused tetanic tension in isolated EDL muscle of the male diabetic group was less as compared to the male control group which was statistically significant (p<0.001). Maximum fused tetanic tension after the fatigue protocol in isolated EDL muscle of the male diabetic group was significantly lower (p<0.001) as compared to the male control group.

Similarly, tetanic tension after 5 minutes of rest period following the fatigue protocol in isolated EDL muscle of the male diabetic group was less as compared to the male control group which was statistically significant (p < 0.001).

DISCUSSION

The present study was designed to evaluate the contractile functions of slow and fast skeletal muscles in streptozotocin (STZ) induced type 1 diabetic male Sprague Dawley rats. At the end of four weeks of study, maximum isometric twitch tension (ITT) in isolated soleus and extensor digitorum longus (EDL) muscles of the male diabetic group was similar to the healthy controls. Skeletal muscle is the predominant tissue for the whole body lipid oxidation, in which up to 90% of energy requirements at rest are derived from fatty acids14. T1DM is associated with an increased accumulation of intramyocellular lipids (IMCL), which might serve as an alternate energy source in the absence of glucose. In addition, in T1DM, intramyocellular lipid accumulation of more than two fold has been observed in the slow soleus muscle as compared to the fast tibialis anterior muscle15.

Based on the results of present study, it is suggested that the diabetic skeletal muscles derive ATP from intramyocellular lipids to generate normal isometric twitch response.

Time to peak twitch tension (TPT) in isolated soleus muscle of the diabetic group was significantly longer (p < 0.001) as compared to the healthy controls. On the contrary, TPT in isolated extensor digitorum longus (EDL) muscle of the male diabetic group was similar to the control group. The characteristic prolongation in time to peak twitch tension in isolated soleus muscle might be explained in part by a change in isomyosin composition of the skeletal muscle in T1DM. This would have resulted in an increase in the number of type I slow-twitch fibers at the expense of type II fast-twitch fibers. Loss of fast isomyosins and appearance of slow isoforms have been demonstrated in a biochemical study on rat's gastrocnemius muscle, 4 weeks after STZ injection16. An impairment of calcium release from the sarcoplasmic reticulum could be another factor for the prolongation in time to peak twitch tension in the diabetic soleus muscle.

Previous morphological studies had provided evidence that sarcoplasmic reticulum and T tubules were disrupted in skeletal and cardiac muscles of the diabetic rats17. In another study, similar prolongation in time to peak twitch tension in soleus muscle of rats was observed after 14 days of STZ induced T1DM.18. Time to peak twitch tension (TPT) was unaffected in extensor digitorum longus muscle in the present study. This reflects that the change in isomyosin form was not of sufficient magnitude to effect the whole muscle measurements, as extensor digitorum longus muscle was still predominantly composed of type IIB or fast-twitch glycolytic fibers.

Time taken to relax to 50% of the peak twitch tension (HRT) in isolated soleus muscle of the male diabetic group was significantly longer (p < 0.001) as compared to the control group. On the contrary, HRT in isolated extensor digitorum longus (EDL) muscle of the male diabetic group was similar to the control group.

In present study, the marked slowing in time taken to relax to 50% of the peak twitch tension in isolated soleus muscle is suggestive of impairment in calcium sequestration by sarcoplasmic reticulum. In vitro studies on heart and skeletal muscles of diabetic rats have demonstrated reduced sarcoplasmic reticulum calcium ATPase activity19. On the other hand, sarcoplasmic reticulum is much more extensive in the fast muscles which might be the reason that a similar level of damage to the soleus muscle did not have the same effect on extensor digitorum longus muscle, because of its greater functional reserve.

Maximum fused tetanic tension in isolated soleus muscle of the male diabetic group was similar to the control group. On the other hand, maximum fused tetanic tension in isolated extensor digitorum longus (EDL) muscle of the male diabetic group was significantly lower (p<0.001) as compared to the control group. This finding highlights the fact that soleus muscle can maintain maximum fused tetanic tension with respect to its muscle mass. This could be associated to the minimal atrophy of type I or slow-twitch oxidative fibers of soleus muscle in T1DM. In a study conducted on 90% pancreatectomized diabetic model of young Sprague Dawley rats, maximum fused tetanic tension was recorded in the gastrocnemius-plantaris-soleus (GPS) muscle complex, after 4 and 8 weeks of diabetes. In that study, maximum fused tetanic tension at 4 weeks was similar between the diabetic (7.95 +- 0.91 N/g) and control (7.86 +- 0.91 N/g) groups20. On the other hand, extensor digitorum longus muscle showed a marked decline in force output.

This could be due to the preferential atrophy of the predominant fast-twitch glycolytic fibers in extensor digitorum longus muscle which have the greatest tension generating capacity21. Hyperglycemia has been associated with profound reduction in protein synthesis and increased protein break down in fast-twitch glycolytic fibers when compared with the slow-twitch fibers22.

Maximum fused tetanic tension after the fatigue protocol in isolated soleus and extensor digitorum longus (EDL) muscles of the male diabetic group was significantly lower (p<0.001) as compared to the control group. The present study reflects that both type I or slow-twitch oxidative fibers and type II or fast-twitch glycolytic fibers exhibit increased fatigability in T1DM. This could be attributed to the common factors affecting type I and type II fibers, such as, reduced ATP production due to the reduced substrate availability, accumulation of metabolic end products that impair contractile events, changes in intracellular and extracellular muscle electrolyte concentration that reduce muscle excitability, and alterations in sarcoplasmic reticulum calcium handling properties23. In a study conducted on male Wistar rats, results similar to the present study were observed.

In that study, maximum fused tetanic tension in soleus muscle showed 32+-3% decline in healthy control rats after 5 minutes of fatigue protocol, whereas, reduction in maximum fused tetanic tension in soleus muscle of the diabetic rats was 52+-2% which was significantly greater (p<0.001). The extensor digitorum longus muscle in diabetic rats also manifested reduction in maximum fused tetanic tension by 72 +- 3% after 5 minutes of fatigue protocol which was significantly greater (p<0.001) than the reduction in tension (63+- 3%) observed in healthy control rats24.

In present study, tetanic tension after 5 minutes of rest period following the fatigue protocol in isolated soleus and extensor digitorum longus (EDL) muscles of the male diabetic group was significantly lower (p<0.001) as compared to the control group. The data of present study highlights that recovery from fatigue is significantly impaired in both type I and type II fibers in T1DM. This could be due to the reduced replenishment of intracellular energy resources (i.e. creatine phosphate, glycogen or ATP), failure of removal of metabolic by products with potential deleterious effects (e.g., H+ and lactate)25, and changes in muscle ion concentration (e.g., Na+, K+, Ca+2, Mg+2)26.

CONCLUSION

It is concluded that streptozotocin induced type 1 diabetes mellitus manifests differential effects on the contractile properties of slow and fast skeletal muscles of male Sprague Dawley rats. In slow muscles, the tetanic tension remains unaffected, while the speed related properties get slowed down. In fast muscles, the tetanic tension is decreased, whereas, the speed related properties remain unaffected. There occurs reduction in resistance to and recovery from fatigue in both slow and fast skeletal muscles.

ACKNOWLEDGEMENTS

We would like to thank the National University of Sciences and Technology (NUST) for the financial assistance to accomplish this original work. Our thanks also go to Dr. Ali Hussain, Miss Irum and Mr. Fawad Fazal for their constant technical assistance throughout the study.

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Publication:Pakistan Armed Forces Medical Journal
Date:Sep 30, 2012
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