Lactic acid restores skeletal muscle force in an in vitro fatigue model: are voltage-gated chloride channels involved?

High interstitial K(+) concentration ([K(+)]) has been reported to impede normal propagation of electrical impulses along the muscle cell membrane (sarcolemma) and then also into the transverse tubule system; this is one considered underlying mechanism associated with the development of muscle fatigue. Interestingly, the extracellular buildup of lactic acid, once considered an additional cause for muscle fatigue, was recently shown to have force-restoring effects in such conditions. Specifically, it was proposed that elevated lactic acid (and intracellular acidosis) may lead to inhibition of voltage-gated chloride channels, thereby reestablishing better excitability of the muscle cell sarcolemma. In the present study, using an in vitro muscle contractile experimental setup to study functionally viable rectus abdominis muscle preparations obtained from normal swine, we examined the effects of 20 mM lactic acid and 512 µM 9-anthracenecarboxylic acid (9-AC; a voltage-gated chloride channel blocker) on the force recovery of K(+)-depressed (10 mM K(+)) twitch forces. We observed a similar muscle contractile restoration after both treatments. Interestingly, at elevated [K(+)], myotonia (i.e., hyperexcitability or afterdepolarizations), usually present in skeletal muscle with inherent or induced chloride channel dysfunctions, was not observed in the presence of either lactic acid or 9-AC. In part, these data confirm previous studies showing a force-restoring effect of lactic acid in high-[K(+)] conditions. In addition, we observed similar restorative effects of lactic acid and 9-AC, implicating a beneficial mechanism via voltage-gated chloride channel modulation.

In vitro effects of propofol and volatile agents on pharmacologically induced chloride channel myotonia.

BACKGROUND: Anesthetic choice for patients with chloride channel myotonia remains under debate. The authors have, therefore, investigated the in vitro effects of various anesthetic agents on pharmacologically induced chloride channel myotonia. METHODS: Functionally viable (> 10 mN force generation) rectus abdominis muscle preparations obtained from normal swine were investigated using in vitro muscle contracture test baths. During continuous 0.1-Hz supramaximal electrical stimulation, the chloride channel blocker 9-anthracenecarboxylic acid (64 microM) was added before the addition of propofol or one of three volatile anesthetics. The concentration of propofol in either Intralipid (n = 11) or dimethyl sulfoxide (n = 10) was doubled every 10 min (from 4-512 microM). The concentration of halothane (n = 8), isoflurane (n = 8), and sevoflurane (n = 8) was doubled from 0.25 vol% up to the maximum dose according to calibrated vaporizers. Control muscle bundles were either untreated (n = 30) or exposed to 9-anthracenecarboxylic acid (n = 19). RESULTS: The myotonic reactions induced by 9-anthracenecarboxylic acid were reversed by high-dose (> 64 microM) propofol (P < 0.01). Halothane, isoflurane, or sevoflurane each enhanced the myotonic reactions at 5.4 (P < 0.001), 0.21 (P < 0.01), and 0.5 minimum alveolar concentrations (P < 0.05), respectively. CONCLUSIONS: The authors' in vitro data imply that propofol administration for general anesthesia may be better suited for patients with chloride channel myotonia versus volatile anesthetics. In isolated swine skeletal muscle bundles, propofol elicited a reversal of 9-anthracenecarboxylic acid-induced chloride channel myotonia, whereas volatile anesthetics further increased the associated myotonic reactions.

Effects of Ablation (Radio Frequency, Cryo, Microwave) on Physiologic Properties of the Human Vastus Lateralis.

OBJECTIVE: Ablative treatments can sometimes cause collateral injury to surrounding muscular tissue, with important clinical implications. In this study, we investigated the changes in muscle physiology of the human vastus lateralis when exposed to three different ablation modalities: radiofrequency ablation, cryoablation, and microwave ablation. METHODS: We obtained fresh vastus lateralis tissue biopsy specimens from nine patients (age range: 29-73 years) who were undergoing in vitro contracture testing for malignant hyperthermia. Using leftover waste tissue, we prepared 46 muscle bundles that were utilized in tissue baths before and after ablation. RESULTS: After ablation with all the three modalities, we noted dose-dependent sustained reductions in peak force (strength of contraction), as well as transient increases in baseline force (resting muscle tension). But, over the subsequent 3-h recovery period, peak force improved and the baseline force consistently recovered to below its preablation levels. CONCLUSION: The novel in vitro methodologies we developed to investigate changes in muscle physiology after ablation can be used to study a spectrum of ablation modalities and also to make head-to-head comparisons of different ablation modalities. SIGNIFICANCE: As the role of ablative treatments continues to expand, our findings provide unique insights into the resulting changes in muscle physiology. These insights could enhance the safety and efficacy of ablations and help individuals design and develop novel medical devices.

Assessment of Ablative Therapies in Swine: Response of Respiratory Diaphragm to Varying Doses.

Ablation is a common procedure for treating patients with cancer, cardiac arrhythmia, and other conditions, yet it can cause collateral injury to the respiratory diaphragm. Collateral injury can alter the diaphragm's properties and/or lead to respiratory dysfunction. Thus, it is important to understand the diaphragm's physiologic and biomechanical properties in response to ablation therapies, in order to better understand ablative modalities, minimize complications, and maximize the safety and efficacy of ablative procedures. In this study, we analyzed physiologic and biomechanical properties of swine respiratory diaphragm muscle bundles when exposed to 5 ablative modalities. To assess physiologic properties, we performed in vitro tissue bath studies and measured changes in peak force and baseline force. To assess biomechanical properties, we performed uniaxial stress tests, measuring force-displacement responses, stress-strain characteristics, and avulsion forces. After treating the muscle bundles with all 5 ablative modalities, we observed dose-dependent sustained reductions in peak force and transient increases in baseline force-but no consistent dose-dependent biomechanical responses. These data provide novel insights into the effects of various ablative modalities on the respiratory diaphragm, insights that could enable improvements in ablative techniques and therapies.

Freeze-thaw induced biomechanical changes in arteries: role of collagen matrix and smooth muscle cells.

Applications involving freeze-thaw, such as cryoplasty or cryopreservation can significantly alter artery biomechanics including an increase in physiological elastic modulus. Since artery biomechanics plays a significant role in hemodynamics, it is important to understand the mechanisms underlying these changes to be able to help control the biomechanical outcome post-treatments. Understanding of these mechanisms requires investigation of the freeze-thaw effect on arterial components (collagen, smooth muscle cells or SMCs), as well as the components' contribution to the overall artery biomechanics. To do this, isolated fresh swine arteries were subjected to thermal (freeze-thaw to -20 degrees C for 2 min or hyperthermia to 43 degrees C for 2 h) and osmotic (0.1-0.2 M mannitol) treatments; these treatments preferentially altered either the collagen matrix (hydration/stability) or smooth muscle cells (SMCs), respectively. Tissue dehydration, thermal stability and SMC functional changes were assessed from bulk weight measurements, analyses of the thermal denaturation profiles using Fourier transform infrared (FTIR) spectroscopy and in vitro arterial contraction/relaxation responses to norepinephrine (NE) and acetylcholine (AC), respectively. Additionally, Second Harmonic Generation (SHG) microscopy was performed on fresh and frozen-thawed arteries to directly visualize the changes in collagen matrix following freeze-thaw. Finally, the overall artery biomechanics was studied by assessing responses to uniaxial tensile testing. Freeze-thaw of arteries caused: (a) tissue dehydration (15% weight reduction), (b) increase in thermal stability (approximately 6.4 degrees C increase in denaturation onset temperature), (c) altered matrix arrangement observed using SHG and d) complete SMC destruction. While hyperthermia treatment also caused complete SMC destruction, no tissue dehydration was observed. On the other hand, while 0.2 M mannitol treatment significantly increased the thermal stability (approximately 4.8 degrees C increase in denaturation onset), 0.1 M mannitol treatment did not result in any significant change. Both 0.1 and 0.2 M treatments caused no change in SMC function. Finally, freeze-thaw (506+/-159 kPa), hyperthermia (268+/-132 kPa) and 0.2 M mannitol (304+/-125 kPa) treatments all caused significant increase in the physiological elastic modulus (Eartery) compared to control (185+/-92 kPa) with the freeze-thaw resulting in the highest modulus. These studies suggest that changes in collagen matrix arrangement due to dehydration as well as SMC destruction occurring during freeze-thaw are important mechanisms of freeze-thaw induced biomechanical changes.

Hibernation induction trigger reduces hypoxic damage of swine skeletal muscle.

A link between the cardioprotective benefits of pharmacological preconditioning and natural mammalian hibernation is considered to involve the cellular activation of opioid receptors and subsequent opening of K(ATP) channels. In previous studies, we have demonstrated the protective effects of specific delta-opioid agonists against porcine cardiac ischemia/reperfusion injury. We hypothesize here that preincubation with hibernation induction trigger (HIT) should confer a similar protection in skeletal muscles. Therefore, muscle bundles from swine were pretreated with plasma from hibernating woodchucks (HWP) for 30 min, then exposed to hypoxia for 90 min and reoxygenation for 120 min. Stimulated twitch forces were assessed. The functional effects of pretreatment with nonhibernation (summer) woodchuck plasma, a K(ATP) blocker, or opioid antagonist were also studied. During the reoxygenation period, significantly greater force recoveries were observed only for bundles pretreated with HWP; this response was blocked by naloxone (P < 0.05). We conclude that HIT pretreatment could be used to confer protection against hypoxia/reperfusion injury of skeletal muscles of nonhibernators; it could potentially be utilized to prevent injury during surgical procedures requiring ischemia.

In vitro studies of human hearts.

BACKGROUND: Isolated mammalian hearts have been used in numerous studies that have led to many important discoveries in cardiac physiology, pharmacology, and surgery. Multiple methods of perfusion have been described including retrograde and/or antegrade flows and crystalloid or blood perfusates. Furthermore, multiple species have been utilized for such studies including the following: rat, rabbit, guinea pig, canine, and swine. The objective of this study was to describe a unique isolated heart preparation, utilizing human hearts not viable for transplant, which allows for physiologic perfusion and endocardial imaging. METHODS: Utilizing standard cardiac transplantation procedures, 12 human hearts deemed not viable for transplant were explanted to an isolated heart apparatus. A clear, modified Krebs-Henseleit buffer was used as a blood substitute, which allowed for endocardial imaging utilizing 6.0 mm endoscopic video cameras inserted into the cardiac chambers. The hearts were perfused in Langendorff (retrograde) and/or working (physiologic) mode. RESULTS: Eleven of 12 hearts achieved the following performance in working mode: peak left ventricular pressure of 21.5 to 75.8 mm Hg, with an average of 42.7 +/- 19.9 mm Hg. Intracardiac anatomical imaging was possible in all hearts, providing unique views of normal and pathological endocardial anatomy as well as biomedical device-heart interactions. CONCLUSIONS: We have described a unique isolated heart preparation with which we have successfully reanimated 11 human hearts deemed not viable for transplant, perfused them by working mode, and performed intracardiac anatomical imaging. This approach provides a novel means for obtaining images of functional human cardiac anatomy and various types of unique biomedical assessments.