Background/Aims: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3... more Background/Aims: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na+/K+ pump/ATPase [NKP, NKA], Na+-K+-2Cl+ [NKCC] and K+-Cl- [KCC] cotransporters, and channel-supported K+ loss) in human lens epithelial cells [LECs]. Methods: K+ loss and K+ uptake, using Rb+ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl- replacement by NO3ˉ to determine KCC. 3H-Ouabain binding was performed on a pig renal NKA in the presence and absence of CET. Bcl-2 protein and NKA sequences were aligned and motifs identified and mapped using PROSITE in conjunction with BLAST alignments and analysis of conservation and structural similarity based on prediction of secondary and crystal structures. Results: CET inhibited NKP and NKCC by \u3e90% (IC50 values ∼35 and ∼15 µM, respectively) ...
The highly conserved, cardiotonic steroid binding site (also termed ouabain binding site) on the ... more The highly conserved, cardiotonic steroid binding site (also termed ouabain binding site) on the primary α subunit of Na,K-ATPase plays a receptor signaling role in a range of vital cell processes and is a therapeutic target for human disease. Mouse lines with altered affinity for cardiotonic steroids on the α1 or α2 subunit isoform of Na,K-ATPase, without any change in pump activity, were developed by the late Jerry B Lingrel and are a valuable tool for studying its physiological roles and drug actions. In one model, the normally ouabain resistant α1 isoform was rendered sensitive to ouabain binding. In a second model, the normally sensitive α2 isoform was rendered resistant to ouabain binding. Additional useful models are obtained by mating these mice. To further advance their use, we developed a rapid, real-time PCR method that detects mutant alleles using specific primers and fluorescent probes. PCR is performed in fast mode with up to 15 samples processed in 40 min. The method ...
Regeneration of adult skeletal muscle after injury is coordinated by complex interactions between... more Regeneration of adult skeletal muscle after injury is coordinated by complex interactions between the injured muscle and the innate immune system. Myeloid lineage cells predominate in this process. This study examined the role of Krüppel like factor 2 (KLF2), a zinc-finger transcription factor that regulates myeloid cell activation state, in muscle regeneration. Gastrocnemius muscles of wild-type and myeKlf2-/- mice, which lack KLF2 in all myeloid cells, were subjected to cardiotoxin injury and followed for 21 days. Injured muscles of myeKlf2-/- contained more infiltrating, inflammatory Ly6C+ monocytes, with elevated expression of inflammatory mediators. Infiltrating monocytes matured earlier into pro-inflammatory macrophages with phenotype Ly6C+, CD11b+, F4/80+. Inflammation resolved earlier and progressed to myogenesis, marked by an earlier decline of Ly6C+ macrophages and their replacement with anti-inflammatory Ly6C- populations, in association with elevated expression of factor...
ABSTRACTWe developed a sensitive, ratiometric method to measure simultaneously 13C-labeled glucos... more ABSTRACTWe developed a sensitive, ratiometric method to measure simultaneously 13C-labeled glucose and rubidium in biological samples using ICP-MS. The method uses probe-assisted ultra-sonication with water to extract 13C-[6C]-labeled-D-glucose and other polar analytes from mammalian tissues. It extracts >80% of the reference value for Rb and >95 % of 13C in a CRM spiked with 13C-[6C]-labeled-D-glucose in the micro-molar range. Using optimized instrument conditions, the method achieves a stable 13C/12C signal without spectral interferences. The 13C/12C signal is independent of sample composition and depends linearly on the concentration of 13C-[6C]-labeled-D-glucose in spiked samples. Overall, the method achieves a limit of detection of 10 µM for 6-C-labeled 13C glucose in biological tissues. This detection capability for carbon in biological matrices by ICP-MS opens a wider range of applications for ICP-MS in biomedical research. As proof-of-principle, we combined 13C detecti...
The potassium affinities of Na,K-ATPase isozymes are important determinants of their physiologica... more The potassium affinities of Na,K-ATPase isozymes are important determinants of their physiological roles in skeletal muscle. This study measured the apparent K+ and Rb+ affinities of the Na,K-ATPase α1 and α2 isozymes in intact, dissociated myofibers obtained from WT and genetically altered mice (α1S/Sα2R/R and skα2−/−). It also validates a new method to quantify cations in intact, dissociated myofibers, using inductively coupled plasma mass spectrometry (ICP-MS). Our findings were that: (1) The extracellular substrate sites of Na,K-ATPase bind Rb+ and K+ with comparable apparent affinities; however; turnover rate is reduced when Rb+ is the transported ion; (2) The rate of Rb+ uptake by the Na,K-ATPase is not constant but declines with a half-time of approximately 1.5 min; (3) The apparent K+ affinity of the α2 isozymes for K+ is significantly lower than α1. When measured in intact fibers of WT and α1S/Sα2R/R mice in the presence of 10 µM ouabain; the K1/2,K of α1 and α2 isozymes ar...
Background: Skeletal muscle myopathy and exercise intolerance are diagnostic hallmarks of heart f... more Background: Skeletal muscle myopathy and exercise intolerance are diagnostic hallmarks of heart failure (HF). However, the molecular adaptations of skeletal muscles during dilated cardiomyopathy (DCM)-mediated HF are not completely understood. Methods: Skeletal muscle structure and function were compared in wild-type (WT) and cardiac myosin binding protein-C null mice (t/t), which develop DCM-induced HF. Cardiac function was examined by echocardiography. Exercise tolerance was measured using a graded maximum treadmill running test. Hindlimb muscle function was assessed in vivo from measurements of plantar flexor strength. Inflammatory status was evaluated from the expression of inflammatory markers and the presence of specific immune cell types in gastrocnemius muscles. Muscle regenerative capacityat days 3, 7, and 14 after eccentric contraction-induced injury was determined from the number of phenotypically new and adult fibers in the gastrocnemius, and functional recovery of plantar flexion torque. Results: t/t mice developed DCM-induced HF in association with profound exercise intolerance, consistent with previous reports. Compared to WT, t/t mouse hearts show significant hypertrophy of the atria and ventricles and reduced fractional shortening, both systolic and diastolic. In parallel, the skeletal muscles of t/t mice exhibit weakness and myopathy. Compared to WT, plantar flexor muscles of t/t null mice produce less peak isometric plantar torque (Po), develop torque more slowly (+ dF/dt), and relax more slowly (− dF/dt, longer half-relaxation times,1/2RT). Gastrocnemius muscles of t/t mice have a greater number of fibers with smaller diameters and central nuclei. Oxidative fibers, both type I and type IIa, show significantly smaller cross-sectional areas and more central nuclei. These fiber phenotypes suggest ongoing repair and regeneration under homeostatic conditions. In addition, the ability of muscles to recover and regenerate after acute injury is impaired in t/t mice. Conclusions: Our studies concluded that DCM-induced HF induces a unique skeletal myopathy characterized by decreased muscle strength, atrophy of oxidative fiber types, ongoing inflammation and damage under homeostasis, and impaired regeneration after acute muscle injury. Furthermore, this unique myopathy in DCM-induced HF likely contributes to and exacerbates exercise intolerance. Therefore, efforts to develop therapeutic interventions to treat skeletal myopathy during DCM-induced HF should be considered.
Summary Skeletal muscle repair and regeneration after injury requires coordinated interactions be... more Summary Skeletal muscle repair and regeneration after injury requires coordinated interactions between the innate immune system and the injured muscle. Myeloid cells predominate in these interactions. This study examined the role of KLF2, a zinc-finger transcription factor that regulates immune cell activation, in specifying myeloid cell functions during muscle regeneration. Loss of KLF2 in myeloid lineage cells (myeKlf2−/− mice) dramatically enhanced the initial inflammatory response to acute muscle injury (cardiotoxin). Injured muscles showed dramatically elevated expression of inflammatory mediators and greater numbers of infiltrating, pro-inflammatory monocytes that matured earlier into activated macrophages. Notably, the inflammatory phase resolved earlier and regeneration progressed to myogenesis, marked by elevated expression of factors that promote the formation of new fibers from satellite cells. Regeneration was completed earlier, with phenotypically normal adult fibers integrated into the muscle syncytium. These findings identify myeloid KLF2 as a key regulator of myeloid cell functions in adult skeletal muscle regeneration.
American journal of physiology. Endocrinology and metabolism, 2018
The distribution of Na/K-ATPase α-isoforms in skeletal muscle is unique, with α1 as the minor (15... more The distribution of Na/K-ATPase α-isoforms in skeletal muscle is unique, with α1 as the minor (15%) isoform and α2 comprising the bulk of the Na/K-ATPase pool. The acute and isoform-specific role of α2 in muscle performance and resistance to fatigue is well known, but the isoform-specific role of α1 has not been as thoroughly investigated. In vitro, we reported that α1 has a role in promoting cell growth that is not supported by α2. To assess whether α1 serves this isoform-specific trophic role in the skeletal muscle, we used Na/K-ATPase α1-haploinsufficient (α1) mice. A 30% decrease of Na/K-ATPase α1 protein expression without change in α2 induced a modest yet significant decrease of 10% weight in the oxidative soleus muscle. In contrast, the mixed plantaris and glycolytic extensor digitorum longus weights were not significantly affected, likely because of their very low expression level of α1 compared with the soleus. The soleus mass reduction occurred without change in total Na/K...
American journal of physiology. Cell physiology, Jan 8, 2017
Marked loss of skeletal muscle mass occurs under various conditions of disuse, but the molecular ... more Marked loss of skeletal muscle mass occurs under various conditions of disuse, but the molecular and cellular mechanisms leading to atrophy are not completely understood. We investigate early molecular events which might play a role in skeletal muscle remodeling during mechanical unloading (disuse). The effects of acute (6 - 12 h) hindlimb suspension on the soleus muscles from adult rats were examined. The integrity of plasma membrane lipid rafts was tested utilizing cholera toxin B subunit, or fluorescent sterols. In addition, resting intracellular Ca(2+) level was analyzed. Acute disuse disturbed the plasma membrane lipid-ordered phase throughout the sarcolemma and was more pronounced in junctional membrane regions. Ouabain (1 µM), which specifically inhibits the Na,K-ATPase α2 isozyme in rodent skeletal muscles, produced similar lipid rafts changes in control muscles, but was ineffective in suspended muscles, which show an initial loss of α2 Na,K-ATPase activity. Lipid rafts were...
The Na,K-ATPase is essential for the contractile function of skeletal muscle, which expresses the... more The Na,K-ATPase is essential for the contractile function of skeletal muscle, which expresses the α1 and α2 subunit isoforms of Na,K-ATPase. The α2 isozyme is predominant in adult skeletal muscles and makes a greater contribution in working compared with noncontracting muscles. Hindlimb suspension (HS) is a widely used model of muscle disuse that leads to progressive atrophy of postural skeletal muscles. This study examines the consequences of acute (6-12 h) HS on the functioning of the Na,K-ATPase α1 and α2 isozymes in rat soleus (disused) and diaphragm (contracting) muscles. Acute disuse dynamically and isoform-specifically regulates the electrogenic activity, protein, and mRNA content of Na,K-ATPase α2 isozyme in rat soleus muscle. Earlier disuse-induced remodeling events also include phospholemman phosphorylation as well as its increased abundance and association with α2 Na,K-ATPase. The loss of α2 Na,K-ATPase activity results in reduced electrogenic pump transport and depolariz...
The use of ICP-MS to measure metal ion content in biological tissues offers a highly sensitive me... more The use of ICP-MS to measure metal ion content in biological tissues offers a highly sensitive means to study metal-dependent physiological processes. Here we describe the application of ICP-MS to measure membrane transport of Rb and K ions by the Na,K-ATPase in mouse skeletal muscles and human red blood cells. The ICP-MS method provides greater precision and statistical power than possible with conventional tracer flux methods. The method is widely applicable to studies of other metal ion transporters and metal-dependent processes in a range of cell types and conditions.
This study examined the developmental expression of Na,K-ATPase a-and b-subunit isoforms in diffe... more This study examined the developmental expression of Na,K-ATPase a-and b-subunit isoforms in different skeletal muscles of the mouse, and the relationship of Na,K-ATPase a 2 isoform expression to the developing transverse tubules (t-tubules). We measured Na,K-ATPase and dihydropyridine receptor (DHPR) mRNA and protein in the diaphragm and hindlimb muscles from embryonic day 18.5 (E18.5) to 6 weeks postnatal, using DHPR expression to mark the timing of ttubule formation. The Na,K-ATPase subunits showed developmental age-dependent and muscle-specific expression that was controlled by both transcriptional and post-transcriptional mechanisms. The a 1 isoform is expressed at more constant levels in both diaphragm and hindlimb muscles, while the a 2 and b 2 isoforms increase postnatally and show greater muscle variation. b 1 is the sole expressed b-subunit in the diaphragm throughout development, and in the hindlimb muscles at birth. The Na,K-ATPase a 2 subunit is expressed during development when the t-tubules form. These results suggest that the a 2 isoform may serve, in part, a physiological role in the muscle t-tubules.
The effects of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3&#3... more The effects of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) on slow calcium currents (ICa) were investigated using the Vaseline-gap voltage-clamp technique in bullfrog skeletal muscle cut fibers. Both cAMP and cGMP induced a pronounced increase in the amplitude of ICa when applied to the cut ends of fibers. Both cyclic nucleotides also decreased time to peak current at all membrane potentials. The current-voltage relationship was shifted toward more negative potentials by cAMP as well as cGMP. The potentiating effects of cAMP and cGMP on ICa were additive. 8-Bromo analogues of both nucleotides had similar effects on ICa. The beta-adrenergic agonist isoproterenol, applied extracellularly, also produced an increase in the amplitude of ICa and produced a leftward shift in the current-voltage relationship. These results suggest that both cAMP and cGMP modulate calcium slow channels in bullfrog skeletal muscle fibers, causing stimulation of the ICa. The effect of cyclic nucleotides on ICa in bullfrog skeletal muscle contrasts with that in mammalian cardiac muscle, in which the same nucleotides produce opposite effects on the slow ICa, i.e., in cardiac muscle cAMP stimulates, and cGMP inhibits, the slow ICa.
Background/Aims: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3... more Background/Aims: Chelerythrine [CET], a protein kinase C [PKC] inhibitor, is a prop-apoptotic BH3-mimetic binding to BH1-like motifs of Bcl-2 proteins. CET action was examined on PKC phosphorylation-dependent membrane transporters (Na+/K+ pump/ATPase [NKP, NKA], Na+-K+-2Cl+ [NKCC] and K+-Cl- [KCC] cotransporters, and channel-supported K+ loss) in human lens epithelial cells [LECs]. Methods: K+ loss and K+ uptake, using Rb+ as congener, were measured by atomic absorption/emission spectrophotometry with NKP and NKCC inhibitors, and Cl- replacement by NO3ˉ to determine KCC. 3H-Ouabain binding was performed on a pig renal NKA in the presence and absence of CET. Bcl-2 protein and NKA sequences were aligned and motifs identified and mapped using PROSITE in conjunction with BLAST alignments and analysis of conservation and structural similarity based on prediction of secondary and crystal structures. Results: CET inhibited NKP and NKCC by \u3e90% (IC50 values ∼35 and ∼15 µM, respectively) ...
The highly conserved, cardiotonic steroid binding site (also termed ouabain binding site) on the ... more The highly conserved, cardiotonic steroid binding site (also termed ouabain binding site) on the primary α subunit of Na,K-ATPase plays a receptor signaling role in a range of vital cell processes and is a therapeutic target for human disease. Mouse lines with altered affinity for cardiotonic steroids on the α1 or α2 subunit isoform of Na,K-ATPase, without any change in pump activity, were developed by the late Jerry B Lingrel and are a valuable tool for studying its physiological roles and drug actions. In one model, the normally ouabain resistant α1 isoform was rendered sensitive to ouabain binding. In a second model, the normally sensitive α2 isoform was rendered resistant to ouabain binding. Additional useful models are obtained by mating these mice. To further advance their use, we developed a rapid, real-time PCR method that detects mutant alleles using specific primers and fluorescent probes. PCR is performed in fast mode with up to 15 samples processed in 40 min. The method ...
Regeneration of adult skeletal muscle after injury is coordinated by complex interactions between... more Regeneration of adult skeletal muscle after injury is coordinated by complex interactions between the injured muscle and the innate immune system. Myeloid lineage cells predominate in this process. This study examined the role of Krüppel like factor 2 (KLF2), a zinc-finger transcription factor that regulates myeloid cell activation state, in muscle regeneration. Gastrocnemius muscles of wild-type and myeKlf2-/- mice, which lack KLF2 in all myeloid cells, were subjected to cardiotoxin injury and followed for 21 days. Injured muscles of myeKlf2-/- contained more infiltrating, inflammatory Ly6C+ monocytes, with elevated expression of inflammatory mediators. Infiltrating monocytes matured earlier into pro-inflammatory macrophages with phenotype Ly6C+, CD11b+, F4/80+. Inflammation resolved earlier and progressed to myogenesis, marked by an earlier decline of Ly6C+ macrophages and their replacement with anti-inflammatory Ly6C- populations, in association with elevated expression of factor...
ABSTRACTWe developed a sensitive, ratiometric method to measure simultaneously 13C-labeled glucos... more ABSTRACTWe developed a sensitive, ratiometric method to measure simultaneously 13C-labeled glucose and rubidium in biological samples using ICP-MS. The method uses probe-assisted ultra-sonication with water to extract 13C-[6C]-labeled-D-glucose and other polar analytes from mammalian tissues. It extracts >80% of the reference value for Rb and >95 % of 13C in a CRM spiked with 13C-[6C]-labeled-D-glucose in the micro-molar range. Using optimized instrument conditions, the method achieves a stable 13C/12C signal without spectral interferences. The 13C/12C signal is independent of sample composition and depends linearly on the concentration of 13C-[6C]-labeled-D-glucose in spiked samples. Overall, the method achieves a limit of detection of 10 µM for 6-C-labeled 13C glucose in biological tissues. This detection capability for carbon in biological matrices by ICP-MS opens a wider range of applications for ICP-MS in biomedical research. As proof-of-principle, we combined 13C detecti...
The potassium affinities of Na,K-ATPase isozymes are important determinants of their physiologica... more The potassium affinities of Na,K-ATPase isozymes are important determinants of their physiological roles in skeletal muscle. This study measured the apparent K+ and Rb+ affinities of the Na,K-ATPase α1 and α2 isozymes in intact, dissociated myofibers obtained from WT and genetically altered mice (α1S/Sα2R/R and skα2−/−). It also validates a new method to quantify cations in intact, dissociated myofibers, using inductively coupled plasma mass spectrometry (ICP-MS). Our findings were that: (1) The extracellular substrate sites of Na,K-ATPase bind Rb+ and K+ with comparable apparent affinities; however; turnover rate is reduced when Rb+ is the transported ion; (2) The rate of Rb+ uptake by the Na,K-ATPase is not constant but declines with a half-time of approximately 1.5 min; (3) The apparent K+ affinity of the α2 isozymes for K+ is significantly lower than α1. When measured in intact fibers of WT and α1S/Sα2R/R mice in the presence of 10 µM ouabain; the K1/2,K of α1 and α2 isozymes ar...
Background: Skeletal muscle myopathy and exercise intolerance are diagnostic hallmarks of heart f... more Background: Skeletal muscle myopathy and exercise intolerance are diagnostic hallmarks of heart failure (HF). However, the molecular adaptations of skeletal muscles during dilated cardiomyopathy (DCM)-mediated HF are not completely understood. Methods: Skeletal muscle structure and function were compared in wild-type (WT) and cardiac myosin binding protein-C null mice (t/t), which develop DCM-induced HF. Cardiac function was examined by echocardiography. Exercise tolerance was measured using a graded maximum treadmill running test. Hindlimb muscle function was assessed in vivo from measurements of plantar flexor strength. Inflammatory status was evaluated from the expression of inflammatory markers and the presence of specific immune cell types in gastrocnemius muscles. Muscle regenerative capacityat days 3, 7, and 14 after eccentric contraction-induced injury was determined from the number of phenotypically new and adult fibers in the gastrocnemius, and functional recovery of plantar flexion torque. Results: t/t mice developed DCM-induced HF in association with profound exercise intolerance, consistent with previous reports. Compared to WT, t/t mouse hearts show significant hypertrophy of the atria and ventricles and reduced fractional shortening, both systolic and diastolic. In parallel, the skeletal muscles of t/t mice exhibit weakness and myopathy. Compared to WT, plantar flexor muscles of t/t null mice produce less peak isometric plantar torque (Po), develop torque more slowly (+ dF/dt), and relax more slowly (− dF/dt, longer half-relaxation times,1/2RT). Gastrocnemius muscles of t/t mice have a greater number of fibers with smaller diameters and central nuclei. Oxidative fibers, both type I and type IIa, show significantly smaller cross-sectional areas and more central nuclei. These fiber phenotypes suggest ongoing repair and regeneration under homeostatic conditions. In addition, the ability of muscles to recover and regenerate after acute injury is impaired in t/t mice. Conclusions: Our studies concluded that DCM-induced HF induces a unique skeletal myopathy characterized by decreased muscle strength, atrophy of oxidative fiber types, ongoing inflammation and damage under homeostasis, and impaired regeneration after acute muscle injury. Furthermore, this unique myopathy in DCM-induced HF likely contributes to and exacerbates exercise intolerance. Therefore, efforts to develop therapeutic interventions to treat skeletal myopathy during DCM-induced HF should be considered.
Summary Skeletal muscle repair and regeneration after injury requires coordinated interactions be... more Summary Skeletal muscle repair and regeneration after injury requires coordinated interactions between the innate immune system and the injured muscle. Myeloid cells predominate in these interactions. This study examined the role of KLF2, a zinc-finger transcription factor that regulates immune cell activation, in specifying myeloid cell functions during muscle regeneration. Loss of KLF2 in myeloid lineage cells (myeKlf2−/− mice) dramatically enhanced the initial inflammatory response to acute muscle injury (cardiotoxin). Injured muscles showed dramatically elevated expression of inflammatory mediators and greater numbers of infiltrating, pro-inflammatory monocytes that matured earlier into activated macrophages. Notably, the inflammatory phase resolved earlier and regeneration progressed to myogenesis, marked by elevated expression of factors that promote the formation of new fibers from satellite cells. Regeneration was completed earlier, with phenotypically normal adult fibers integrated into the muscle syncytium. These findings identify myeloid KLF2 as a key regulator of myeloid cell functions in adult skeletal muscle regeneration.
American journal of physiology. Endocrinology and metabolism, 2018
The distribution of Na/K-ATPase α-isoforms in skeletal muscle is unique, with α1 as the minor (15... more The distribution of Na/K-ATPase α-isoforms in skeletal muscle is unique, with α1 as the minor (15%) isoform and α2 comprising the bulk of the Na/K-ATPase pool. The acute and isoform-specific role of α2 in muscle performance and resistance to fatigue is well known, but the isoform-specific role of α1 has not been as thoroughly investigated. In vitro, we reported that α1 has a role in promoting cell growth that is not supported by α2. To assess whether α1 serves this isoform-specific trophic role in the skeletal muscle, we used Na/K-ATPase α1-haploinsufficient (α1) mice. A 30% decrease of Na/K-ATPase α1 protein expression without change in α2 induced a modest yet significant decrease of 10% weight in the oxidative soleus muscle. In contrast, the mixed plantaris and glycolytic extensor digitorum longus weights were not significantly affected, likely because of their very low expression level of α1 compared with the soleus. The soleus mass reduction occurred without change in total Na/K...
American journal of physiology. Cell physiology, Jan 8, 2017
Marked loss of skeletal muscle mass occurs under various conditions of disuse, but the molecular ... more Marked loss of skeletal muscle mass occurs under various conditions of disuse, but the molecular and cellular mechanisms leading to atrophy are not completely understood. We investigate early molecular events which might play a role in skeletal muscle remodeling during mechanical unloading (disuse). The effects of acute (6 - 12 h) hindlimb suspension on the soleus muscles from adult rats were examined. The integrity of plasma membrane lipid rafts was tested utilizing cholera toxin B subunit, or fluorescent sterols. In addition, resting intracellular Ca(2+) level was analyzed. Acute disuse disturbed the plasma membrane lipid-ordered phase throughout the sarcolemma and was more pronounced in junctional membrane regions. Ouabain (1 µM), which specifically inhibits the Na,K-ATPase α2 isozyme in rodent skeletal muscles, produced similar lipid rafts changes in control muscles, but was ineffective in suspended muscles, which show an initial loss of α2 Na,K-ATPase activity. Lipid rafts were...
The Na,K-ATPase is essential for the contractile function of skeletal muscle, which expresses the... more The Na,K-ATPase is essential for the contractile function of skeletal muscle, which expresses the α1 and α2 subunit isoforms of Na,K-ATPase. The α2 isozyme is predominant in adult skeletal muscles and makes a greater contribution in working compared with noncontracting muscles. Hindlimb suspension (HS) is a widely used model of muscle disuse that leads to progressive atrophy of postural skeletal muscles. This study examines the consequences of acute (6-12 h) HS on the functioning of the Na,K-ATPase α1 and α2 isozymes in rat soleus (disused) and diaphragm (contracting) muscles. Acute disuse dynamically and isoform-specifically regulates the electrogenic activity, protein, and mRNA content of Na,K-ATPase α2 isozyme in rat soleus muscle. Earlier disuse-induced remodeling events also include phospholemman phosphorylation as well as its increased abundance and association with α2 Na,K-ATPase. The loss of α2 Na,K-ATPase activity results in reduced electrogenic pump transport and depolariz...
The use of ICP-MS to measure metal ion content in biological tissues offers a highly sensitive me... more The use of ICP-MS to measure metal ion content in biological tissues offers a highly sensitive means to study metal-dependent physiological processes. Here we describe the application of ICP-MS to measure membrane transport of Rb and K ions by the Na,K-ATPase in mouse skeletal muscles and human red blood cells. The ICP-MS method provides greater precision and statistical power than possible with conventional tracer flux methods. The method is widely applicable to studies of other metal ion transporters and metal-dependent processes in a range of cell types and conditions.
This study examined the developmental expression of Na,K-ATPase a-and b-subunit isoforms in diffe... more This study examined the developmental expression of Na,K-ATPase a-and b-subunit isoforms in different skeletal muscles of the mouse, and the relationship of Na,K-ATPase a 2 isoform expression to the developing transverse tubules (t-tubules). We measured Na,K-ATPase and dihydropyridine receptor (DHPR) mRNA and protein in the diaphragm and hindlimb muscles from embryonic day 18.5 (E18.5) to 6 weeks postnatal, using DHPR expression to mark the timing of ttubule formation. The Na,K-ATPase subunits showed developmental age-dependent and muscle-specific expression that was controlled by both transcriptional and post-transcriptional mechanisms. The a 1 isoform is expressed at more constant levels in both diaphragm and hindlimb muscles, while the a 2 and b 2 isoforms increase postnatally and show greater muscle variation. b 1 is the sole expressed b-subunit in the diaphragm throughout development, and in the hindlimb muscles at birth. The Na,K-ATPase a 2 subunit is expressed during development when the t-tubules form. These results suggest that the a 2 isoform may serve, in part, a physiological role in the muscle t-tubules.
The effects of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3&#3... more The effects of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) on slow calcium currents (ICa) were investigated using the Vaseline-gap voltage-clamp technique in bullfrog skeletal muscle cut fibers. Both cAMP and cGMP induced a pronounced increase in the amplitude of ICa when applied to the cut ends of fibers. Both cyclic nucleotides also decreased time to peak current at all membrane potentials. The current-voltage relationship was shifted toward more negative potentials by cAMP as well as cGMP. The potentiating effects of cAMP and cGMP on ICa were additive. 8-Bromo analogues of both nucleotides had similar effects on ICa. The beta-adrenergic agonist isoproterenol, applied extracellularly, also produced an increase in the amplitude of ICa and produced a leftward shift in the current-voltage relationship. These results suggest that both cAMP and cGMP modulate calcium slow channels in bullfrog skeletal muscle fibers, causing stimulation of the ICa. The effect of cyclic nucleotides on ICa in bullfrog skeletal muscle contrasts with that in mammalian cardiac muscle, in which the same nucleotides produce opposite effects on the slow ICa, i.e., in cardiac muscle cAMP stimulates, and cGMP inhibits, the slow ICa.
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Papers by Judith Heiny