MHC Isoform Expression Research Articles

MiR-183 and miR-96 orchestrate both glucose and fat utilization in skeletal muscle.

Our knowledge of the coordination of fuel usage in skeletal muscle is incomplete. Whether and how microRNAs are involved in the substrate selection for oxidation is largely unknown. Here we show that mice lacking miR-183 and miR-96 have enhanced muscle oxidative phenotype and altered glucose/lipid homeostasis. Moreover, loss of miR-183 and miR-96 results in a shift in substrate utilization toward fat relative to carbohydrates in mice. Mechanistically, loss of miR-183 and miR-96 suppresses glucose utilization in skeletal muscle by increasing PDHA1 phosphorylation via targeting FoxO1 and PDK4. On the other hand, loss of miR-183 and miR-96 promotes fat usage in skeletal muscle by enhancing intramuscular lipolysis via targeting FoxO1 and ATGL. Thus, our study establishes miR-183 and miR-96 as master coordinators of fuel selection and metabolic homeostasis owing to their capability of modulating both glucose utilization and fat catabolism. Lastly, weshow that loss of miR-183 and miR-96 can alleviate obesity andimprove glucose metabolism in high-fat diet-induced mice, suggesting that miR-183 and miR-96 may serve as therapeutic targets for metabolic diseases.

Extensive diversification of MHC in Chinese giant salamanders Andrias davidianus (Anda-MHC) reveals novel splice variants

A series of MHC alleles (including 26 class IA, 27 class IIA, and 17 class IIB) were identified from Chinese giant salamander Andrias davidianus (Anda-MHC). These genes are similar to classical MHC molecules in terms of characteristic domains, functional residues, deduced tertiary structures and genetic diversity. The majority of variation between alleles is found in the putative peptide-binding region (PBR), which is driven by positive Darwinian selection. The coexistence of two isoforms in MHC IA, IIA, and IIB alleles are shown: one full-length transcript and one novel splice variant. Despite lake of the external domains, these variants exhibit similar subcellular localization with the full-length transcripts. Moreover, the expression of MHC isoforms are up-regulated upon in vivo and in vitro stimulation with Andrias davidianus ranavirus (ADRV), suggesting their potential roles in the immune response. The results provide insights into understanding MHC variation and function in this ancient and endangered urodele amphibian.

Effect of Estrogen on Molecular and Functional Characteristics of the Rodent Vaginal Muscularis

Vaginal atrophy is a consequence of menopause; however, little is known concerning the effect of a decrease in systemic estrogen on vaginal smooth muscle structure and function. As the incidence of pelvic floor disorders increases with age, it is important to determine if estrogen regulates the molecular composition and contractility of the vaginal muscularis. The goal of this study was to determine the effect of estrogen on molecular and functional characteristics of the vaginal muscularis utilizing a rodent model of surgical menopause. Three- to 4-month old Sprague-Dawley rats underwent sham laparotomy (Sham, N = 18) or ovariectomy (Ovx, N = 39). Two weeks following surgery, animals received a subcutaneous osmotic pump containing vehicle (Sham, Ovx) or 17β-estradiol (Ovx). Animals were euthanized 1 week later, and the proximal vagina was collected for analysis of contractile protein expression and in vitro studies of contractility. Measurements were analyzed using a one-way analysis of variance followed by Tukey's post hoc analysis (α = 0.05). Protein and mRNA transcript expression levels of contractile proteins, in vitro measurements of vaginal contractility. Ovariectomy decreased the expression of carboxyl-terminal myosin heavy chain isoform (SM1) and h-caldesmon and reduced the amplitude of contraction of the vaginal muscularis in response to KCl. Estradiol replacement reversed these changes. No differences were detected in the % vaginal muscularis, mRNA transcript expression of amino-terminal MHC isoforms, l-caldesmon expression, and maximal velocity of shortening. Systemic estrogen replacement restores functional and molecular characteristics of the vaginal muscularis of ovariectomized rats. Our results indicate that menopause is associated with changes in the vaginal muscularis, which may contribute to the increased incidence of pelvic floor disorders with age.

A Novel Method to Measure Glucose Uptake and Myosin Heavy Chain Isoform Expression of Single Fibers From Rat Skeletal Muscle

Skeletal muscle includes many individual fibers with diverse phenotypes. A barrier to understanding muscle glucose uptake at the cellular level has been the absence of a method to measure glucose uptake by single fibers from mammalian skeletal muscle. This study’s primary objective was to develop a procedure to measure glucose uptake by single fibers from rat skeletal muscle. Rat epitrochlearis muscles were incubated ex vivo with [3H]-2-deoxy-d-glucose, with or without insulin or AICAR, before isolation of ~10–30 single fibers from each muscle. Fiber type (myosin heavy chain [MHC] isoform) and glucose uptake were determined for each single fiber. Insulin-stimulated glucose uptake (which was cytochalasin B inhibitable) varied according to MHC isoform expression, with ~2-fold greater values for IIA versus IIB or IIX fibers and ~1.3-fold greater for hybrid (IIB/X) versus IIB fibers. In contrast, AICAR-stimulated glucose uptake was ~1.5-fold greater for IIB versus IIA fibers. A secondary objective was to assess insulin resistance of single fibers from obese versus lean Zucker rats. Genotype differences were observed for insulin-stimulated glucose uptake and inhibitor κB (IκB)-β abundance in single fibers (obese less than lean), with decrements for glucose uptake (44–58%) and IκB-β (25–32%) in each fiber type. This novel method creates a unique opportunity for future research focused on understanding muscle glucose uptake at the cellular level.

Effect of estrogen on molecular and functional characteristics of the rodent vaginal muscularis

Vaginal atrophy is a consequence of menopause, however little is known concerning the effect of estrogen on vaginal smooth muscle function. The goal of this study was to determine the effect of estrogen on molecular and functional characteristics of the vaginal muscularis using a rodent model of surgical menopause. 3–4 month old Sprague Dawley rats underwent sham laparotomy (Sham) or ovariectomy (Ovx). Two weeks following surgery, animals received a subcutaneous osmotic pump containing vehicle (Sham, Ovx) or 17‐ β estradiol (Ovx). Animals were euthanized one week later and the proximal vagina was collected for molecular and in vitro contractility studies. Measurements were analyzed using one way ANOVA followed by post hoc analysis. Ovariectomy decreased 1) mRNA transcript (P<0.05) and protein (P < 0.001) expression of carboxyl‐terminal myosin heavy chain isoform (MHC), SM1, 2) protein expression of h‐caldesmon (P < 0.01) and 3) peak force of vaginal strips in response to KCl (P < 0.001). Estradiol replacement reversed these changes. No differences were detected in the % vaginal muscularis, amino terminal MHC isoform expression and maximal velocity of shortening. Our results suggest that menopause is associated with changes in the vaginal muscularis which may contribute to the increased incidence of pelvic floor disorders with age.Funding: AUA Research Scholar Award, Maureen Basha

A Novel Method to Measure Glucose Uptake by Single Skeletal Muscle Fibers Reveals a Similar Level of Insulin Resistance for Type IIA, IIB, IIX and IIB/X Fibers from Obese Zucker Rat Epitrochlearis Muscle

We have previously developed a method to measure glucose uptake (GU) by single fibers from rat skeletal muscle, showing in normal Wistar rat muscle, insulin‐stimulated GU was significantly greater in fibers expressing IIA vs. all other measured fiber types (IIA> IIB, IIX, IIB/X). Our aim for this study was to use this method to gain insights into insulin resistance. For this purpose we measured glucose uptake by single fibers from Obese Zucker (OZ; a widely used model of insulin resistance) vs. Lean Zucker (LZ) rats. Epitrochlearis muscles were incubated ex vivo with [3H]‐2‐ deoxy‐D‐glucose, with or without insulin, before isolation of ~10–30 single fibers from each muscle. Fiber type (myosin heavy chain, MHC, isoform) and GU were determined for each fiber. Basal GU values by single fibers were not significantly different between OZ and LZ, regardless of MHC isoform expression. For GU by single fibers from insulin‐stimulated muscles, there was a significant main effect of genotype, and post‐hoc analysis indicated that insulin‐stimulated GU values for each fiber type were significantly greater for LZ vs. OZ rats. However, the calculated relative decrement for insulin‐stimulated GU between fibers from OZ vs. LZ did not differ significantly among the fiber types (IIA= 44%, IIB=58%, IIX=52%, IIB/X =52%). These results provide unique information about skeletal muscle GU for a fuller understanding insulin resistance.

Abnormalities of satellite cells function in amyotrophic lateral sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is characterized by progressive denervation leading to muscle atrophy prevented, during the early phase, by compensatory reinnervation. Little is known about muscle fibre regeneration capacity in ALS. We have carried out in vivo and in vitro investigation of skeletal muscle in ALS. Seven ALS patients underwent a deltoid muscle biopsy. Immunohistochemical analysis revealed various degrees of denervation- and reinnervation-related changes in the ALS muscle biopsies including satellite cells (SCs) activation and regenerating fibres. Only 3/7 primary cultures of ALS muscle cells were successfully established and had sufficient myogenicity, as assessed by desmin positivity, to be used without further purification. This was in contrast with the cultures derived from control muscles, predominantly desmin-positive cells. Although capable to proliferate in vitro, ALS-derived SCs presented an abnormal senescent-like morphology. Markers of senescence, including senescent-associated (SA)-βGal activity and p16 expression, were increased. Furthermore, ALS-derived SCs were also unable to fully differentiate in vitro as shown by abnormal myotubes morphology and reduced MHC isoform expression, compared to control myotubes. Our study suggests that SC function is altered in ALS. This could limit the efficacy of compensatory processes and therefore could contribute to the progression of muscle atrophy and weakness.

116 Cardiac remodeling and failure late after myocardial infarction is exacerbated by tissue hypothyroidism

Previous studies have shown that thyroid hormone (TH) signaling is altered after acute myocardial infarction or myocardial hypertrophy with potential physiological consequences. We investigated whether such changes are related to the severity of cardiac dysfunction. Myocardial infarction was induced in rats by coronary artery ligation (AMI), while SHAM operated animals served as controls (SHAM, n=8). Both AMI and SHAM hearts were studied after 34 weeks. AMI were divided to heart failure (AMI-HF, n=6) and non-heart failure group (AMI-NHF, n=7) as assessed by the ratio of lung weight to body weight (LGW/BW) and right ventricle weight to body weight (RV/BW). Contractile function and left ventricular (LV) remodeling was assessed by echocardiography. TRα1 was increased in both border and remote regions of viable LV of AMI-NHF (2.0 fold and 1.8 fold), while it decreased in AMI-HF (2.0 fold and 1.5 fold) vs SHAM, p<0.05. TRβ1 was reduced in border and remote regions in AMI-HF (2.8 fold and 2.0 fold) and AMI-NHF (3.6 fold and 2.0 fold) vs SHAM, p<0.05. T3 and T4 were not different between groups. Accordingly, this response corresponded to changes in cardiac function (table) and in MHC isoform expression. A marked increase in β-MHC was observed in both border and remote regions of LV of AMI-HF (76% and 75%), p<0.05 vs both SHAM and AMI-NHF while a small but significant increase in β-MHC was found in both border and remote regions of LV in AMI-NHF (62% and 60%) as compared to SHAM (48% and 50%). LGW/BW RV/BW LVEDD (mm) LVEDS (mm) EF% SHAM 3.77 (0.15) 0.35 (0.02) 6.9 (0.15) 4.0 (0.2) 75.6 (2.3) AMI-HF 3.95 (0.4) 0.43 (0.02) 8.8 (0.22) * 7.1 (0.3) * 41.2 (2.6) AMI-NHF 6.90 (0.8) ** 0.82 (0.1) ** 10.4 (0.50) ** 9.2 (0.5) ** 27 (2.0) Post-infarct remodelling results in tissue hypothyroidism. The occurrence of heart failure is accompanied by distinct changes in TR and MHC isoform expression corresponding to a marked regression to the fetal pattern. * p<0.05 vs SHAM, ** p<0.05 vs SHAM and AMI-HF

Cardiac impairment in rabbits fed a high-fat diet is counteracted by dehydroepiandrosterone supplementation

Aims The biochemical and structural cardiac oxidative-dependent damage induced by high-fat (HF) diet was examined in a rabbit model, together with the role of dehydroepiandrosterone (DHEA) in contrasting tissue damage. Main methods New Zealand white rabbits fed a HF diet supplemented or not with DHEA (0.02%) were utilized for 12 weeks. Oxidative stress, inflammatory and necrosis parameters, fatty deposition, heavy-chain myosin isoforms (MHC) expression and papillary muscle functionality were examined in the left ventricle of rabbits. Key findings Rabbits fed a HF diet that showed hyperglycemia, insulin resistance and dyslipidemia together with increase of oxidative stress and of advanced end-glycation product levels have been observed. Concerning pro-inflammatory insults, there was increased p65-NFkB activation and increased tumor necrosis factor-alpha and C-reactive protein expressions. Cellular damage induced by the HF diet was detected through the switch of expression of MHC isoforms, indicating impairment of cardiac contractility, confirmed by altered of basal parameters of papillary muscle functionality. Rabbits fed the HF diet supplemented with DHEA showed a partial reduction of oxidative stress and the inflammatory state. Cardiac necrosis, the shift of MHC isoforms, and cardiac functionality, were also partially counteracted. Significance Rabbits fed with a HF diet showed a beneficial effect when low-dose DHEA was added to the diet. The steroid, without affecting high plasma glucose level or insulin resistance, restored oxidative balance, lowered lipid levels and inflammation insults, preventing cellular and functional alterations of cardiac tissue and thus delaying the onset of cardiac damage.

ALT-C, a disintegrin-like Cys-rich protein from Bothrops alternatus, increases skeletal myoblast viability

ALT-C, an ECD motif (glutamic acid, cysteine, aspartic acid) disintegrin from Bothrops alternatus snake venom, induces α2β1 integrin-mediated signaling and neutrophil chemotaxis. In vitro, in human umbilical vein endothelial cells (HUVEC), ALT-C induces cell proliferation, thus showing an interesting potential for tissue regeneration studies. This work aimed to evaluate the influence of ALT-C in myoblast viability and differentiation. Myoblasts were obtained from hind limb muscles of 3 to 4-day old Wistar rats. The cells were incubated with ALT-C at different concentrations and incubation periods were followed by total RNA isolation. cDNA synthesis and real time polymerase chain reaction (PCR) were performed with primers of myoD as well as of both (slow and fast) myosin heavy chain isoforms (MHC). ECD-disintegrin increased myoblast viability in a dose-dependent way, mostly with 50 to 100 nM concentrations, and such effect was more prevalent after 48 hours. No changes in gene expression of both MHC isoforms were observed in ALT-C-treated cells. MyoD expression was not detected, which suggests that myoblasts were in mature stages. Protease activity and cytokine array tested in a medium of 50 nM ALT-C-treated cells after 48 hours were not different from controls. In conclusion, it was shown that myoblats are sensitive to ALT-C indicating an integrin-mediated intracellular signaling that increases cell viability.

Enzyme‐ and immunohistochemical aspects of skeletal muscle fibers in brown bear (Ursus arctos)

To further elucidate the pattern of MHC isoform expression in skeletal muscles of large mammals, in this study the skeletal muscles of brown bear, one of the largest mammalian predators with an extraordinary locomotor capacity, were analyzed. Fiber types in longissimus dorsi, triceps brachii caput longum, and rectus femoris muscles were determined according to the myofibrillar ATPase (mATPase) histochemistry and MHC isoform expression, revealed by a set of antibodies specific to MHC isoforms. The oxidative (SDH) and glycolytic enzyme (alpha-GPDH) capacity of fibers was demonstrated as well. By mATPase histochemistry five fiber types, i.e., I, IIC, IIA, IIAX, IIX were distinguished. Analyzing the MHC isoform expression, we assume that MHC-I, -IIa, and -IIx are expressed in the muscles of adolescent bears. MHC-I isoform was expressed in Type-I fibers and coexpressed with presumably -IIa isoform, in Type-IIC fibers. Surprisingly, two antibodies specific to rat MHC-IIa stained those fast fibers, that were histochemically and immunohistochemically classified as Type IIX. This assumption was additionally confirmed by complete absence of fiber staining with antibody specific to rat MHC-IIb and all fast fiber staining with antibody that according to our experience recognizes MHC-IIa and -IIx of rat. Furthermore, quite high-oxidative capacity of all fast fiber types and their weak glycolytic capacity also imply for MHC-IIa and -IIx isoform expression in fast fibers of bear. However, in adult, full-grown animal, only MHC-I and MHC-IIa isoforms were expressed. The expression of only two fast isoforms in bear, like in many other large mammals (humans, cat, dog, goat, cattle, and horse) obviously meets the weight-bearing and locomotor demands of these mammals.

Differential contribution of cardiac sarcomeric proteins in the myofibrillar force response to stretch

The present study examined the contribution of myofilament contractile proteins to regional function in guinea pig myocardium. We investigated the effect of stretch on myofilament contractile proteins, Ca(2+) sensitivity, and cross-bridge cycling kinetics (K (tr)) of force in single skinned cardiomyocytes isolated from the sub-endocardial (ENDO) or sub-epicardial (EPI) layer. As observed in other species, ENDO cells were stiffer, and Ca(2+) sensitivity of force at long sarcomere length was higher compared with EPI cells. Maximal K (tr) was unchanged by stretch, but was higher in EPI cells possibly due to a higher alpha-MHC content. Submaximal Ca(2+)-activated K (tr) increased only in ENDO cells with stretch. Stretch of skinned ENDO muscle strips induced increased phosphorylation in both myosin-binding protein C and myosin light chain 2. We concluded that transmural MHC isoform expression and differential regulatory protein phosphorylation by stretch contributes to regional differences in stretch modulation of activation in guinea pig left ventricle.

Activation of SIRT1, a class III histone deacetylase, contributes to fructose feeding-mediated induction of the α-myosin heavy chain expression

Fructose feeding has been shown to induce the cardiac alpha-myosin heavy chain (MHC) expression and protect the heart from ischemia- and reperfusion-mediated cell injury. This study was designed to investigate the mechanism involved in the effect of this sugar on MHC gene expression and cardiac protection. Adult mice were fed with a 6-propyl-2-thiouracil (PTU) diet or PTU combined with a fructose-rich diet. PTU treatment made animals hypothyroid and that resulted in total replacement of cardiac alpha-MHC with the beta-MHC isoform. Addition of fructose in the PTU diet led to reexpression of the alpha-MHC isoform to a significant level. Similar induction of alpha-MHC expression was also seen when PTU diet was combined with resveratrol, an agonist of sirtuin (SIRT) 1 deacetylase. Analysis of heart lysate of these animals indicated that fructose feeding augmented the NAD-to-NADH ratio and the cardiac SIRT1 levels, thus suggesting a role of SIRT1 in fructose-mediated activation of alpha-MHC isoform. To analyze a direct effect of SIRT1 on MHC isoform expression, we generated transgenic mice expressing SIRT1 in the heart. Treatment of these transgenic mice with PTU diet did not lead to disappearance of alpha-MHC, as it did in the nontransgenic animals. SIRT1 overexpression also activated the alpha-MHC gene promoter in transient transfection assays, thus confirming a role of SIRT1 in the induction of alpha-MHC expression. Fructose feeding also attenuated the MHC isoform shift and blocked the cardiac hypertrophy response associated with pressure overload, which was again associated with the induction of cardiac SIRT1 levels. These results demonstrate that fructose feeding protects the heart by induction of the SIRT1 deacetylase and highlight its role in the induction of alpha-MHC gene expression.

Thyroid hormone is required for the phenotype transitions induced by the pharmacological inhibition of calcineurin in adult soleus muscle of rats

The present experiment was designed to examine the effects of hypothyroidism and calcineurin inhibition induced by cyclosporin A (CsA) administration on both contractile and metabolic soleus muscle phenotypes, with a novel approach to the signaling pathway controlling mitochondrial biogenesis. Twenty-eight rats were randomly assigned to four groups, normothyroid, hypothyroid, and orally treated with either CsA (25 mg/kg, N-CsA and H-CsA) or vehicle (N-Vh and H-Vh), for 3 wk. Muscle phenotype was estimated by the MHC profile and activities of oxidative and glycolytic enzymes. We measured mRNA levels of the peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), the major regulator of mitochondrial content. We also studied the expression of the catalytic A-subunit of calcineurin (CnA) both at protein and transcript levels and mRNA levels of modulatory calcineurin inhibitor proteins (MCIP)-1 and -2, which are differentially regulated by calcineurin activity and thyroid hormone, respectively. CsA-administration induced a slow-to-fast MHC transition limited to the type IIA isoform, which is associated with increased oxidative capacities. Hypothyroidism strongly decreased both the expression of fast MHC isoforms and oxidative capacities. Effects of CsA administration on muscle phenotype were blocked in conditions of thyroid hormone deficiency. Changes in the oxidative profile were strongly related to PGC-1 alpha changes and associated with phosphorylation of p38 MAPK. Calcineurin and MCIPs mRNA levels were decreased by both hypothyroidism and CsA without additive effects. Taken together, these results suggest that adult muscle phenotype is primarily under the control of thyroid state. Physiological levels of thyroid hormone are required for the effects of calcineurin inhibition on slow oxidative muscle phenotype.

Paradoxical Effects Of Endurance Training And Chronic Hypoxia On Soleus Myofibrillar ATPase Activity

PURPOSE: The aim of this study was to determine the changes in soleus myofibrillar ATPase (m-ATPase) activity and MHC isoform expression after endurance training and/or chronic hypoxic exposure. METHODS: Dark Agouti rats were randomly divided into four groups: control, normoxic sedentary (N, n = 6); normoxic trained (NT, n = 6); hypoxic sedentary (H, n = 6) and hypoxic trained (HT, n = 6) group. Rats lived and trained in normoxia at 760 mmHg (N and NT), or hypobaric hypoxia at 550 mmHg; (≊ 2800 m) (H and HT). Training consisted of 5 sessions/week of treadmill run at 80% of maximal aerobic velocity (MAV) for five weeks. MAV was reached when the rat was not able to follow the incremen increase in speed imposed by the driven wheel and turned in the wheel more than three times. M-ATPase activity was measured by Rapid Flow Quench technique; myosin subunits were analyzed with mono- and two-dimensional gel electrophoresis. RESULTS: Endurance training significantly increased m-ATPase (P < 0.05), although no significant change in MHC isoform occurred in normoxia, there was a fast-to-slow shift in MHC isoform in hypoxia. Chronic hypoxia depressed m-ATPase (P < 0.05) in spite of a slow-to-fast transition in MHC isoform distribution. Indeed, the rate constants of m-ATPase (kF; s-1) for N and NT are respectively 0.032 ± 0.002 s-1 and 0.046 ± 0.003 s-1, while for H and HT 0.031 ±0.001 s-1 and 0.034 ± 0.004 s-1. CONCLUSION: Dissociation between variations in m-ATPase and changes in MHC isoform expression were observed. Changes in fraction of active myosin heads, in myosin light chain isoform (MLC) distribution or in MLC phosphorylation were not sufficient to explain the variations in m-ATPase. Myosin post-translational modifications or changes in other myofibrillar proteins may therefore be responsible for the observed variations in m-ATPase activity.

Differences in the Expression of MHC Isoforms in Single Muscle Fibers After A18 wk Cold Adaptaionin Hamsters

PURPOSE: The purpose of this study was to determine alterations in the expression of MHC isoforms in single muscle fibers after a 18 wk cold exposure in hamsters. METHODS: Ten Golden hamsters (Mesocrietus auratus), aged 15∼17 weeks were randomly assigned to two groups: control group (n=5, CO) and cold exposed group (n=5, CE). The CE group was exposed to cold temperature at 5°C±1 for 18 weeks while the CO group was housed for 18 weeks at 22°C±2. Electrophoretic analyses were performed in single muscle fibers of the vastus intermeidus (VI) muscle (n=50 per muscle) as well as in whole VI muscle. RESULTS: Significant increases in body weight, heart weight, heart rate and body temperature were observed in CE group compared to CO group. Interestingly, blood glucose was significantly lowered in CE as compared to CO (42.2±18.6 vs. 97.4±22.2mg/ dL). MHC type IIA disappeared completely in whole muscle (VI) in CE whereas CO possessed 26% in type IIA. However, CE showed significant increases in MHC type IIX and type IIB as compared to CO. As for single fiber analyses, co-expression of MHC type IIA and IIX was significantly increased in CO, but type IIX and IIB was significantly highly co-expressed in CE, suggesting that type IIX is a dominant isoform in CE. CONCLUSIONS: It is plausible that the transformed MHC isoform type IIX is a muscle adaptation to cold environment, yields metabolic efficiency.

Adaptation and maladaptation of the heart in diabetes: Part II: potential mechanisms.

The prevailing concept of the heart’s response to changes in its environment is a complex network of inter-connecting signal transduction cascades.1 In such a scheme, the focus is on communication of various cell surface receptors, heterotrimeric G-proteins, protein kinases, and transcription factors.2–4⇓⇓ Diabetes is a disorder of metabolic dysregulation. At first glance it appears that metabolism and the metabolic consequences of diabetes do not fit into this signal-response coupling scheme. Two questions arise. First, is metabolism simply an “effect” rather than a “cause” of adaptation? Second, is metabolism only a by-product of signal transduction-induced adaptation, allowing equilibrium (and therefore maintenance of function) in the presence of the other adaptational responses? An alternative is to take a new, less restricted view of metabolism. Beyond its stereotypical function as a provider of ATP, alterations in metabolic flux within the cell create essential signals for the adaptation of the heart to situations such as diabetes. This concept is novel for the heart, but has already been considered in the liver. Like the phosphorylation events occurring in signal transduction cascades, changes in metabolic flux are extremely rapid. For example, translocation of GLUT4 to the cell surface in response to insulin occurs within a second.5 We have previously found that increases or decreases in workload also change metabolic fluxes in seconds.6,7⇓ Therefore, changes in metabolites are rapid enough to allow them to act as signaling molecules. Many of these acute changes in metabolic flux are brought about by the same signal transduction cascades believed to be involved in the adaptation of the heart to changes in its environment. Phosphatidylinositol 3-kinase, Ca2+, and protein kinase C, all of which play a role in cardiac adaptation, regulate metabolism in the heart.8,9⇓ Metabolic signals therefore provide a …

Myosin heavy chain isoform expression and Ca2 +-stimulated ATPase activity in single fibres of toad rectus abdominis muscle.

Segments of single fibres from the rectus abdominis (RA) muscles of adult and juvenile cane toads (Bufo marinus) were examined for myosin heavy chain (mHC) isoform expression and Ca2+-stimulated MgATPase activity. mHC isoform analyses were carried out using the recently developed alanine-SDS-PAGE method, which separates one tonic (BmHCT) and three twitch (BmHC1, BmHC2, BmHC3) mHC isoforms in toad skeletal muscle. Ca2+-stimulated MgATPase activity was measured by spectrophotometric determination of Pi, under conditions in which the ATPase associated with the sarcoplasmic reticulum (SR ATPase) was suppressed by feedback inhibition. The mHC-based fibre types identified in this study include three pure twitch fibre types (t1, t2 and t3), expressing BmHC1, BmHC2 or BmHC3 respectively, and seven hybrid fibre types co-expressing a combination of two or three twitch and tonic or twitch and twitch mHC isoforms. The fibre populations dissected from juvenile and adult toad muscles contained 49.4% (juvenile) and 73.7% (adult) mHC hybrids. The average values for Ca2+-stimulated MgATPase in pure twitch fibres and in fibres expressing predominantly (> or = 95%) the tonic mHC isoform (Tp fibres) differed significantly (P < 0.05) from each other and decreased in the order t1 > t2 > t3 > Tp. We conclude that (i) in RA muscles of both juvenile and adult cane toads there is a large proportion of mHC hybrids, some of which co-express twitch and tonic mHC isoforms and (ii) ATPase activities associated with the four mHC isoforms expressed in toad skeletal muscles decrease in the order BmHC1 > BmHC2 > BmHC3 > BmHCT.

Cardiac dysfunction caused by myocardium-specific expression of a mutant thyroid hormone receptor.

Thyroid hormone deficiency has profound effects on the cardiovascular system, resulting in decreased cardiac contractility, adrenergic responsiveness, and vascular volume and increased peripheral vascular resistance. To determine the importance of direct cardiac effects in the genesis of hypothyroid cardiac dysfunction, the cardiac myocyte was specifically targeted with a mutant thyroid hormone receptor (TR)-beta (Delta337T-TR-beta(1)) driven by the alpha-myosin heavy chain (alpha-MHC) gene promoter. As a control in these experiments, a wild-type (Wt) TR-beta(1) was also targeted to the heart by using the same promoter. Transgenic mice expressing the mutant TR displayed an mRNA expression pattern consistent with cardiac hypothyroidism, even though their peripheral thyroid hormone levels were normal. When these animals were rendered hypothyroid or thyrotoxic, mRNA expression of MHC isoforms remained unchanged in the hearts of the Delta337T transgenic animals, in contrast to Wt controls or transgenic animals expressing Wt TR-beta(1), which exhibited the expected changes in steady-state MHC mRNA levels. Studies in Langendorff heart preparations from mutant TR-beta(1) transgenic animals revealed evidence of heart failure with a significant reduction in +dP/dT, -dP/dT, and force-frequency responses compared with values in Wt controls and transgenic mice overexpressing the Wt TR-beta(1). In contrast, in vivo measures of cardiac performance were similar between Wt and mutant animals, indicating that the diminished performance of the mutant transgenic heart in vitro was compensated for by other mechanisms in vivo. This is the first demonstration indicating that isolated cardiac hypothyroidism causes cardiac dysfunction in the absence of changes in the adrenergic or peripheral vascular system.

Expression of atrial myosin light chains but not alpha-myosin heavy chains is correlated in vivo with increased ventricular function in patients with hypertrophic obstructive cardiomyopathy.

The adult rodent heart adapts to increased work load by reexpression of its fetal genes, for example, beta-myosin heavy chain (MHC), in order to improve contractile function. However, the human ventricle regulates contractility by expression of atrial essential myosin light chain (ALC-1) rather than beta-MHC. We evaluated the impact of both mechanisms in patients with hypertrophic cardiomyopathy. MHC isoform expression was quantified at the mRNA and protein levels by reverse transcriptase polymerase chain reaction and immunoblotting, respectively. Although alpha-MHC mRNA was detected in control and hypertrophied human ventricular tissue, alpha-MHC protein was not observed. Similarly, we investigated the expression of ALC-1 by two-dimensional polyacrylamide gel electrophoresis and the clinical and hemodynamic parameters of the patients with hypertrophic cardiomyopathy. We found a significant positive correlation between ALC-1 protein expression and dP/dtmax in the hypertrophied human ventricle in vivo. Correlations between dP/dtmax and expression of protein for the ryanodine receptor and L-type Ca2+ channel were excluded. Our data suggest that reexpression of ALC-1 improves the contractile state of the adult human heart. We propose that two evolutionarily divergent compensatory mechanisms for increased work demand exist in the mammalian heart: MHC regulation in rodents and essential MLC regulation, of cardiac contractility, in humans.