COMPARISON OF CONTRACTILE PROPERTIES BETWEEN SLOW AND FAST SKELETAL MUSCLES OF FEMALE DIABETIC SPRAGUE-DAWLEY RATS.
Objective: To compare isometric contraction, force-frequency relationship and muscle fatigue between slow and fast muscles of female type 2 diabetes mellitus Sprague-Dawley rats.
Study Design: Experimental study.
Place and Duration of Study: Physiology Department, Army Medical College, Rawalpindi and National Institute of Health, Islamabad from Jan to Dec 2015.
Material and Methods: Twenty healthy female Sprague Dawley rats were divided into 2 groups with 10 rats in each group. Group-I (control) was fed with normal diet and group-II (diabetic) was given high fat diet. Group-II was given intraperitoneal streptozotocin (STZ) (35mg/kg body weight) on 15th day. Body weight, blood glucose and TG: HDL ratio were estimated on 21st day to confirm type II diabetes mellitus (T2DM) induction. Soleus and extensor digitorum longus (EDL) muscles were removed intact and fixed in organ bath system containing Krebs Ringer buffer solution and connected to data acquisition unit (iWorx) to study their contractile parameters.
Results: Isometric twitch tensions of slow (soleus) and fast (EDL) muscles were similar in diabetic and control rats. Contraction and half relaxation times were slower in diabetic soleus muscles in comparison to control muscles. Diabetic soleus and EDL muscles displayed significantly (p16.65 mmol/l or 301 mg/dl) and triglyceride and high density lipoprotein ratio (TG:HDL) >1.8 were observed which confirmed the development of T2DM in diabetic group (table-I)4. Afterwards, rats were anaesthetized by ether inhalation4 and their slow and fast muscles (that is soleus and EDL) were removed intact. Their distal tendons were tied by non-absorbable surgical silk and fixed with a support while proximal tendons were tied to the force transducer (FT-100) connected to iWorx advanced animal/human physiology data acquisition unit (AHK/214)5. Whole muscle was mounted in a 25 ml organ bath system containing Krebs-Ringer buffer solution.
It was continuously bubbled with a mixture of 95% O2 (oxygen) and 5% CO2 (carbon dioxide). Temperature of 30degC was maintained by a thermostat6.
Muscles were stimulated using single (1Hz) twitch stimulations with 1 minute rest periods to record isometric twitches, time to peak twitch tension (TPTT) and time taken to relax to 50% of its peak twitch tension or half relaxation time (HRT)7. Force-frequency relationship was determined by recording the tension produced after stimulating the muscle at increasing frequencies (10 to 110 Hz) for 1 second followed by rest of 3 minutes in between. The maximum tetanic force was calculated. Muscle fatigue was induced and recorded after stimulating the muscle with a one second optimum tetanic stimulation every 5 seconds for 5 minutes. A measure of recovery from fatigue was made by recording the tetanic tension after 5 minutes rest period following the fatigue protocol. All muscle tensions were expressed as Newton/gram (N/g) wet muscle mass6. Statistical analysis of data was done by using SPSS version 21. Independent samples t test was applied and a p-value <0.05 was considered significant.
Table-III: Comparison of contractile properties of isolated EDL (fast) muscles of diabetic and control groups.
###Control group###Diabetic group
Contractile properties of EDL muscle###p value
Maximum isometric twitch tension (N/g)###1.89 +- 0.82###2.16 +- 0.89###0.492
Time to peak twitch tension (ms)###1.55 +- 0.52###1.91 +- 0.10###0.058
Half relaxation time (ms)###1.12 +- 0.50###1.27 +- 0.29###0.429
Maximum fused tetanic tension (N/g)###0.33 +- 0.33###0.08 +- 0.05###0.044*
Maximum fused tetanic tension after fatigue
###0.11 +- 0.09###0.01 +- 0.01###0.016*
Tetanic tension after 5 minutes of rest period
###0.02 +- 0.01###0.004 +- 0.01###0.019*
following fatigue protocol (N/g)
The body weight, plasma glucose and CPK of all rats were within normal range initially. On 21st day T2DM induction was confirmed based on increased blood glucose levels and TG:HDL ratio (table-I). Body weight of diabetic group was increased significantly (p=0.001).
There was no change in isometric twitch tension in both soleus i.e. slow (table-II) and EDL i.e. fast (table-III) muscles of diabetic group as compared to their control counterparts. The TPTT and HRT were significantly prolonged in isolated soleus but not in EDL muscles of diabetic group. The maximum fused tetanic tension (MFTT) was significantly reduced (p=0.044) in isolated EDL but not in soleus muscle (p=0.135) of diabetic group. The MFTT after fatigue protocol and tetanic tension after 5 minutes of rest period following fatigue protocol were significantly reduced in both isolated soleus and EDL muscles of diabetic group (tables II and III).
The animal model comprising of feeding high fat diet (HFD) and low dose STZ administration was considered feasible due to its easy access and cost effectiveness8. Srinavasan also recommended a similar animal model for T2DM induction4.
Significant (p= 0.001) increase in body weight of diabetic rats at the end of the study occurred due to the accumulation of intra-myocellular lipid droplets which contributed in the development of insulin resistance in response to HFD intake9. Comparable changes in weight gain had been observed in a study conducted on diabetic male Sprague Dawley rats after feeding HFD10. The diabetic group in our study developed frank hyperglycemia. High fat diet was a contributor and STZ reduced the insulin secretion which caused the development of frank hyperglycemia as it destroyed the b cells of the pancreas. Streptozotocin induced T2DM in Wistar Furth rats produced frank hyperglycemia in female rats (24.6 mmol/l or 443.24 m/dl)11.The higher levels attained in this study could be attributed to the higher dose of STZ (55mg/kg body weight) used.
In a study conducted on diabetic female adult albino Wistar rats, the mean TG:HDL ratio was 2.8412 which is relatively lesser compared to our study (3.26 +- 1.44) probably due to the difference in species of rats or the dose of STZ (40mg/kg) administered.
The MITT of isolated soleus and EDL muscles was similar between diabetic and control groups as it depends on the number of cross linkages between the myosin heads and actin filaments at a given time. These are dependent on ATP and Ca+2 in the sarcoplasm which are sufficient in diabetic muscle for a single twitch. A study on diabetic Wistar rats weighing 200 to 220 gm displayed minimal effect on isometric twitch tension in diabetic rats13.
The TPTT was found significantly longer in isolated soleus muscle but not in EDL muscle of diabetic group. It depends on the efficient release of calcium from sarcoplasmic reticulum which was not affected in fast muscles. The prolonged HRT in soleus muscle is due to increased type 1 fibers leading to a relative reduction in fast fibers especially 2A fibers causing the delay in calcium release14. Similar significant (p<0.05) increase in contraction time was observed in STZ induced T2DM rats10. An insignificant difference in TPTT of fast muscles was reported in a study conducted on T2DM Sprague Dawley rats15.
The HRT of diabetic soleus muscle (table-II) was significantly prolonged while that of EDL was similar to the healthy controls (table-III). A less dense SR in slow muscles caused slower release and uptake of calcium and slower kinetics of contraction2. Similar results were obtained in a study conducted on diabetic Wistar rats whose soleus muscle had a prolonged HRT compared to control group13. Abundant calcium stores in diabetic fast muscles for the single twitch did not alter their HRT. Diabetic rats after T2DM induction did not show significant increase in HRT of diabetic EDL muscles5.
The MFTT was comparable in isolated diabetic soleus muscle but significantly reduced in diabetic EDL muscles. It depends on strongly bound cross bridges with an increased calcium concentration and ATP to generate greater force. The intra-myocellular lipid accumulation in type 1 fibers provides an added source of ATP. Similar observations were recorded in a study conducted on diabetic rats in which MFTT was comparable in diabetic and control groups16. Subject to 12 hours fast, EDL muscles in this study had a reduced muscle glycogen content which was not compensated in the insulin resistant state causing a drop in tetanic tension9. Study conducted on T2DM rats revealed similar significant (p=0.029) lowering of MFTT in diabetic EDL muscles10.
Data suggest significantly increased fatigability in both soleus and EDL muscles of diabetic rats of our study when compared with controls. The diabetic EDL muscles in our study were more fatigued than soleus. Sustained contractions cause reduction in fuel availability in diabetic muscles faster than the controls which impaired the functioning of contractile apparatus leading to their early fatigue and recovery from fatigue was delayed due to their inability to efficiently replenish energy resources17. A study conducted on diabetic rats showed similar lowering of maximum fused tetanic tension after fatigue protocol in rats of chronic diabetes of 2 months duration13. Another study on T2DM Sprague Dawley rats confirmed the increased fatigability in diabetic slow and fast muscles18.
These findings and further studies based on enzymatic analysis of the skeletal muscle fibers can be utilized for improving the management of type 2 diabetic patients and planning exercise regimens for improving muscle function.
In STZ induced type 2 diabetic slow muscles, the tetanic tension remains unaffected while contraction and half relaxation times are longer. In fast muscles, the tetanic tension and the speed related properties remain unaffected. There is reduction in resistance to and recovery from fatigue in both slow and fast skeletal muscles.
CONFLICT OF INTEREST
This study has no conflict of interest to declare by any author.
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|Publication:||Pakistan Armed Forces Medical Journal|
|Date:||Feb 28, 2017|
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