All procedures outlined in this study were approved by the Animal Care Committee of this institution. Sprague Dawley male rats (150–180 g) were anesthetized by inhalation of isoflurane oxygen mixture (2–5% isoflurane in 100% oxygen), intubated, and artificially ventilated. A left thoracotomy between the fourth and fifth ribs was performed, exposing the left ventricular wall. The left coronary artery was ligated. Experiments were performed 6–10 weeks after coronary ligation. Age- and body weight-matched rats served as controls.

Transthoracic echocardiography was performed 1–2 weeks before the experiments. The rats were anesthetized by inhalation of isoflurane oxygen mixture (2–5% isoflurane in 100% oxygen). The transducer was positioned on the left anterior chest, and left ventricular dimensions were measured. The fractional shortening (FS) was determined by echocardiographic measurements. FS is >40% in controls (n = 9) and <30% in HF (n = 10), respectively.

The rats were anesthetized by inhalation of isoflurane oxygen mixture (2–5% isoflurane in 100% oxygen). An endotracheal tube was inserted into the trachea and attached to a ventilator. Polyethylene catheters (PE-50) were inserted into the common carotid artery and external jugular vein for measurement of arterial blood pressure and for fluid administration, respectively. The skin covering the triceps surae muscle and femoral region was surgically separated from the muscle below in order to eliminate inputs from cutaneous afferents in the hindlimb. The sciatic nerve of each leg was isolated and then placed on a stimulating electrode. A needle microprobe connected to a thermometer (Model BAT-12, World Precision Instrument, Sarasota, FL, USA) was directly inserted into the gastrocnemius muscle of the hindlimb to continuously monitor baseline T_m throughout experiments and measure T_m responses to muscle contraction. The animals were ventilated, and tidal CO₂ was monitored by a respiratory gas monitor (Datex-Ohmeda, Madison, WI, USA) and maintained within normal ranges, as previously described (Li et al., 2004a,b; Gao et al., 2007). Body temperature was maintained between 36.5 and 38.5°C by a heating pad and external heat lamps, and fluid balance was stabilized by a continuous infusion of saline.

Decerebration was performed as previously described (Li et al., 2004a,b; Smith et al., 2005; Gao et al., 2007). A transverse section was made anterior to the superior colliculus. Once this procedure was completed, anesthesia was removed from the inhaled mixture. A recovery period of 60 min after decerebration was employed to allow sufficient time for elimination of the effects of anesthesia gas from the preparation.

Arterial blood pressure was measured by connecting the carotid arterial catheter to a pressure transducer (model P23ID, Statham). Mean arterial pressure (MAP) was obtained by integrating the arterial signal with a time constant of 4 s. HR was determined from the arterial pressure pulse. All measured variables were continuously recorded on an eight-channel chart recorder (model TA 4000, Gould, Valley View, OH, USA) and stored on a PC computer that used the PowerLab system (ADInstruments, Castle Hill, Australia). The triceps surae muscle was isolated and the calcaneal bone of the hindlimb was cut. The Achilles tendon was connected to a force transducer for the measurement of muscle tension during electrically induced muscle contraction. The pelvis was stabilized in a spinal unit and the knee joints were secured by clamping the patellar tendon to a spinal unit.

On completion of each experiment, a 2-Fr microMillar pressure transducer catheter (Millar Instruments) was inserted into the right carotid artery and threaded into the left ventricle for measurement of left ventricular end-diastolic pressure (LVEDP). The heart was exercised after intravenous injection of an overdose of sodium pentobarbital (120 mg/kg body weight) followed by 2 ml of a saturated solution of potassium chloride. Wet heart and triceps surae muscle weight were measured. The data collected from rats whose left ventricle FS was <30% were included in MI group of this report.

A 60 min equilibration period was allowed after completion of experimental procedure. Contractions induced by electrical stimulation of the sciatic nerve were then performed in nine healthy control rats and 10 MI rats. Static contraction of the triceps surae muscle was conducted at frequencies of 30 Hz (2.5 times motor threshold and 0.1 ms duration; Gao et al., 2006). Stimulation was sustained for 1 min. Baseline T_m was controlled at 30, 34, and 38°C at a random way, respectively, by using a water-perfused heating pad and an ice bag around the hindlimb muscle. Contraction was performed at different baselines of T_m. There was a 60 min rest period between each bout of contraction. The T_m was measured before, during, and after each of stimulations. Body core temperature and T_m on the contralateral leg were also measured during stimulation.

Arterial blood pressure and developed muscle tension during muscle contraction were recorded on a PC computer that used Power Lab software. T_m was recorded on a temperature monitor. Control values were determined by averaging at least 1 min of the data immediately before the interventions. The peak change of each variable was determined by the peak response from control.

Peak change data for each variable were analyzed with a two-way ANOVA. Changes in temperature during 1 min-stimulation in control rats and MI rats were also analyzed with a two-way ANOVA. Tukey post hoc analyses were utilized to determine differences between groups, as appropriate. All values were expressed mean ± SE. For all analyses, differences were considered significant at P < 0.05. All statistical analyses were performed using SPSS for windows version 13.0.

Following a start of electrical stimulation of the sciatic nerve, T_m increased. The response gradually returned to a steady level after the end of stimulation. At baseline T_m of 30, 34, and 38°C, time courses of contraction-induced increase in T_m during 1 min of contraction are shown in Figures 1A–C. Peak temperature responses to contraction in control rats and MI rats are shown in Figure 1D. It is noted that an increase in T_m was significantly attenuated in MI rats compared with control rats, as resting T_m was 30 and 34°C. However, this effect wasn’t observed, as resting T_m was 38°C. Body core temperature and T_m on the contralateral leg were not significantly changed during stimulation.

Baseline MAP values before contraction were not different in the healthy control animals and the MI rats (Table 3). Electrical stimulation of the sciatic nerve significantly increased MAP in both groups. A greater MAP increase was observed in MI rats, as baseline T_m was 30 and 34°C but not 38°C (Figure 2A). There was no significant difference in HR response to the stimulation (Table 3).

It is noted that there were no significant differences in the muscle tension indicated by time-tension index in the two groups (Figure 2B). This suggests that attenuated T_m and enhanced pressor responses to contraction in the MI rats were not due to development of muscle tension. In addition, absolute muscle tensions appeared to be smaller in MI rats; however, no significant differences were observed in both groups. When baseline T_m was 30, 34, and 38°C, peak tensions were 0.55 ± 0.04, 0.57 ± 0.06, and 0.46 ± 0.04 kg in control; and 0.52 ± 0.03, 0.51 ± 0.02, and 0.44 ± 0.03 kg in MI (P > 0.05, vs. control at all conditions).

Changes in MAP response to static muscle contraction were plotted against contraction-induced increase in T_m. A significant inverse correlation was seen between the pressor response and increased T_m during contraction (Figure 3). Thus, the greater pressor response is likely linked to the lower T_m response in rats with MI.

Evidence supports that ATP sensitive P2X receptors play a role in evoking mechano- and metaboreceptors mediated stimulation of the exercise pressor reflex (Hanna et al., 2002; Li and Sinoway, 2002; Hanna and Kaufman, 2003, 2004). In HF, P2X plays a role in sympathetic abnormalities by demonstrating that the muscle pressor response induced by α, β-me ATP injected into rat hindlimb muscles is exaggerated in HF animals (Gao et al., 2007). Also, P2X receptor-mediated muscle mechanoreceptor contribution to the sympathetic nerve response to exercise is augmented in HF (Li et al., 2004b; Wang et al., 2010). Moreover, the greater response to activation of PX receptor is related to the severity of left ventricular dysfunction (Gao et al., 2007). This may be due to that HF induces upregulation of P2X receptors in the sensory neurons (Gao et al., 2007). However, the intrinsic mechanism responsible for the P2X receptor alternations in rats with HF is unclear.

It has been reported that P2X receptors are temperature-sensitive and P2X activity increases as temperature falls (Garcia-Villalon et al., 1997; Ziganshin et al., 2002; Kluess et al., 2005). The effect of P2X receptor on reflex muscle response has recently been reported to be sensitive to alternations of T_m (Gao et al., 2006). This prior study further suggests that elevated T_m attenuates the pressor response to static muscle contraction.

Result of the present study shows that T_m and pressor responses during contraction tended to be smaller in both healthy and MI rats as baseline T_m was set at 38°C. Moreover, the greater pressor response was linearly related to the lower T_m response. This is consistent with the concept that the effect of P2X receptor on reflex muscle response is sensitive to alternations of T_m and that elevated temperature attenuates the response (Gao et al., 2006). In addition, our data also show that an increase in T_m during contraction was attenuated in HF rats compared with control rats. Thus, the enhanced P2X activity and exaggerated muscle reflex in HF is likely attributed to a lower T_m response.

Metabolite sensitive-TRPV1 receptors have been reported to respond to heating (Wang et al., 2003). In this report, a rise in T_m in contracting muscle was smaller in the MI rats than in the controls. Thus, we postulate that the muscle metaboreflex is blunted by less stimulation of TRPV1 due to lower temperature response in HF. A lower T_m in HF is likely to contribute to the attenuated TRPV1 activities. It has previously been reported that TRPV1 of sensory neurons is downregulated and TRPV1 response is attenuated in HF rats (Li et al., 2004b; Smith et al., 2005; Wang et al., 2010). Data of the present report provide further evidence that suggest that abnormal autonomic adjustments to exercise may be due to abnormalities in T_m response in HF. We believe that in HF mechanoreceptor contribution to muscle sympathetic nerve activity is augmented, whereas metaboreceptor engagement is attenuated. Overall, mechanosensitive afferents may contribute to a greater degree and muscle sympathetic response is exaggerated in HF (Sterns et al., 1991; Middlekauff et al., 2000, 2001; Li et al., 2004b; Momen et al., 2004; Smith et al., 2005).

It is known that T_m rises with exercise (Shellock et al., 1985; Kenny et al., 2003). During exercise, T_m observed in HF patients is abnormal as compared with healthy subjects (Shellock et al., 1985). Cold temperature increases cardiac demand in response to exercise and significantly reduces maximal exercise capacity in patients with HF (Juneau et al., 2002). A further study suggests that warming of exercising legs improves exercise capacity in patients with cardiac disease and low exercise tolerance (Yamanouchi et al., 1996).

An elevation of T_m by heating muscles increases reflex blood pressure and sympathetic nerve responses to exercise and cooling muscle delays those reflex activities (Ray and Gracey, 1997; Ray et al., 1997). It is noted that T_m is increased >4°C and decreased >7°C. When T_m is altered largely, this could directly change sympathetic nervous activity. Also, afferent inputs from skin could be activated in these human studies. This may lead to different patterns of sympathetic responses than pure muscle afferent activation. In contrast, in our current report the skin covering the triceps surae muscle and femoral region was surgically separated from the muscle below, thus we have eliminated inputs from cutaneous afferents in the limb.

It should be noted that in the present report the effects of muscle blood flow on changes of T_m are not determined as muscle contraction is evoked at different temperature conditions. However, previous studies have demonstrated that blood flow directed to exercising muscles is decreased in HF (Lejemtel et al., 1986; Shoemaker et al., 1999), suggesting that blood flow is likely to contribute to attenuated T_m increase in HF as contraction is induced.

Finally, static contraction was induced by the sciatic nerve stimulation in the current study to optimize T_m and pressor responses. A study limitation is that the pressor response induced by the stimulation was possibly due, in part, to direct electrical activation of afferent nerves. However, contraction-induced increase in T_m was unlikely due to the direct stimulation of afferent nerves because core temperature and T_m on the contralateral leg were not altered. Also, the pressor response was closely related to change of T_m during contraction. This is important to address the issue that impaired T_m response can modify the muscle reflex in HF.

In summary, the data of present study demonstrate that contraction-induced increase in T_m is attenuated in HF animals compared with control animals, and the magnitude of the pressor response to contraction is greater as the temperature increase is lower. The impaired temperature response may alter muscle afferent-mediated pressor response via temperature-sensitive P2X and TRPV1 receptors. This investigation provides evidence for the role played by T_m in the exaggerated sympathetic responses to exercise in HF.