Chest compression duration influences outcome between integrated load-distributing band and manual CPR during cardiac arrest.

BACKGROUND: The Circulation Improving Resuscitation Care (CIRC) Trial found equivalent survival in adult out-of-hospital cardiac arrest (OHCA) patients who received integrated load-distributing band CPR (iA-CPR) compared to manual CPR (M-CPR). We hypothesized that as chest compression duration increased, iA-CPR provided a survival benefit when compared to M-CPR. METHODS: A pre-planned secondary analysis of OHCA of presumed cardiac etiology from the randomized CIRC trial. Chest compressions duration was defined as the total number of minutes spent on compressions during resuscitation and identified from transthoracic impedance and accelerometer data recorded by the EMS defibrillator. Logistic regression was used to model the interaction between treatment and duration of chest compressions and was covariate-adjusted for trial site, patient age, witnessed arrest, and initial shockable rhythm. Primary outcome was survival to hospital discharge. RESULTS: We enrolled 4231 subjects and of those, 2012 iA-CPR and 2002 M-CPR had complete outcome and duration of chest compressions data. While covariate-adjusted odds ratio for survival to hospital discharge was 1.86 in favor of iA-CPR (95% CI 1.16-3.0), there was an interaction between duration and study arm. When this was factored into the multivariate equation, the odds ratio for survival to hospital discharge showed a significant benefit for iA-CPR vs. M-CPR for chest compression duration greater than 16.5 min. CONCLUSION: After adjusting for compression duration and duration-treatment interaction, iA-CPR showed a significant benefit for survival to hospital discharge vs. M-CPR in patients with OHCA if chest compression duration was longer than 16.5 min.

Interstitial fluid flow and cyclic strain differentially regulate cardiac fibroblast activation via AT1R and TGF-ß1.

Cardiac fibroblasts are exposed to both cyclic strain and interstitial fluid flow in the myocardium. The balance of these stimuli is affected by fibrotic scarring, during which the fibroblasts transition to a myofibroblast phenotype. The present study investigates the mechanisms by which cardiac fibroblasts seeded in three-dimensional (3D) collagen gels differentiate between strain and fluid flow. Neonatal cardiac fibroblast-seeded 3D collagen gels were exposed to interstitial flow and/or cyclic strain and message levels of collagens type I and III, transforming growth factor ß1 (TGF-ß1), and α-smooth muscle actin (α-SMA) were assessed. Flow was found to significantly increase and strain to decrease expression of myofibroblast markers. Corresponding immunofluorescence indicated that flow and strain differentially regulated α-SMA protein expression. The effect of flow was inhibited by exposure to losartan, an angiotensin II type 1 receptor (AT1R) blocker, and by introduction of shRNA constructs limiting AT1R expression. Blocking of TGF-ß also inhibited the myofibroblast transition, suggesting that flow-mediated cell signaling involved both AT1R and TGF-ß1. Reduced smad2 phosphorylation in response to cyclic strain suggested that TGF-ß is part of the mechanism by which cardiac fibroblasts differentiate between strain-induced and flow-induced mechanical stress. Our experiments show that fluid flow and mechanical deformation have distinct effects on cardiac fibroblast phenotype. Our data suggest a mechanism in which fluid flow directly acts on AT1R and causes increased TGF-ß1 expression, whereas cyclic strain reduces activation of smad proteins. These results have relevance to the pathogenesis and treatment of heart failure.

Human RFamide-related peptide-1 diminishes cellular and integrated cardiac contractile performance.

Peptides influence cardiac dysfunction; however, peptidergic modulation of contractile performance remains relatively uncharacterized. We identified a novel human peptide that modulates mammalian contractile performance. Members of the FMRFamide-related peptide (FaRP) family contain a C-terminal RFamide but structurally variant N-terminal extension. We report human RFamide-related peptide-1 (hRFRP-1) and rat RFRP-1 rapidly and reversibly decreased shortening and relaxation in isolated mammalian cardiac myocytes in a dose dependent manner. The mammalian FaRP, 26RFa, structurally related to RFRP-1 by only an RFamide did not influence myocyte contractile function. The protein kinase C (PKC) inhibitor bisindolylmaleimide-1 blocked hRFRP-1 activity. Pretreatment with pertussis toxin (PTX) did not diminish hRFRP-1 influence on contractile function. In addition, intravenous injection of hRFRP-1 in mice decreased heart rate, stroke volume, ejection fraction, and cardiac output. Collectively these findings are consistent with the conclusion RFRP-1 is an endogenous signaling molecule that activates PKC and acts through a PTX-insensitive pathway to modulate cardiac contractile function. Taken together these negative chronotropic, inotropic, and lusitropic effects of hRFRP-1 are significant; they suggest direct acute cellular and organ-level responses in mammalian heart. This is the first known study to identify a mammalian FaRP with cardio-depressant effects, opening a new area of research on peptidergic modulation of contractile performance. The high degree of RFRP structure conservation from amphibians to mammals, and similarity to invertebrate cardioinhibitory peptides suggests RFRP-1 is involved in important physiological functions. Elucidation of mechanisms involved in hRFRP-1 synthesis, release, and signaling may aid the development of strategies to prevent or attenuate cardiac dysfunction.

Troponin I chimera analysis of the cardiac myofilament tension response to protein kinase A.

Viral-mediated gene transfer of troponin I (TnI) isoforms and chimeras into adult rat cardiac myocytes was used to investigate the role TnI domains play in the myofilament tension response to protein kinase A (PKA). In myocytes expressing endogenous cardiac TnI (cTnI), PKA phosphorylated TnI and myosin-binding protein C and decreased the Ca2+ sensitivity of myofilament tension. In marked contrast, PKA did not influence Ca2+-activated tension in myocytes expressing the slow skeletal isoform of TnI or a chimera (N-slow/card-C TnI), which lack the unique phosphorylatable amino terminal extension found in cTnI. PKA-mediated phosphorylation of a second TnI chimera, N-card/slow-C TnI, which has the amino terminal region of cTnI, caused a decrease in the Ca2+ sensitivity of tension comparable in magnitude to control myocytes. Based on these results, we propose the amino terminal region shared by cTnI and N-card/slow-C TnI plays a central role in determining the magnitude of the PKA-mediated shift in myofilament Ca2+ sensitivity, independent of the isoform-specific functional domains previously defined within the carboxyl terminal backbone of TnI. Interestingly, exposure of permeabilized myocytes to acidic pH after PKA-mediated phosphorylation of cTnI resulted in an additive decrease in myofilament Ca2+ sensitivity. The isoform-specific, pH-sensitive region within TnI lies in the carboxyl terminus of TnI, and the additive response provides further evidence for the presence of a separate domain that directly transduces the PKA phosphorylation signal.

In vivo acceleration of heart relaxation performance by parvalbumin gene delivery.

Defective cardiac muscle relaxation plays a causal role in heart failure. Shown here is the new in vivo application of parvalbumin, a calcium-binding protein that facilitates ultrafast relaxation of specialized skeletal muscles. Parvalbumin is not naturally expressed in the heart. We show that parvalbumin gene transfer to the heart in vivo produces levels of parvalbumin characteristic of fast skeletal muscles, causes a physiologically relevant acceleration of heart relaxation performance in normal hearts, and enhances relaxation performance in an animal model of slowed cardiac muscle relaxation. Parvalbumin may offer the unique potential to correct defective relaxation in energetically compromised failing hearts because the relaxation-enhancement effect of parvalbumin arises from an ATP-independent mechanism. Additionally, parvalbumin gene transfer may provide a new therapeutic approach to correct cellular disturbances in calcium signaling pathways that cause abnormal growth or damage in the heart or other organs.

p53-dependent expression of PIG3 during proliferation, genotoxic stress, and reversible growth arrest.

The p53-inducible gene 3 (PIG3) was recently identified in a screen for genes induced by p53 before the onset of apoptosis. PIG3 shares significant homology with oxidoreductases from several species. In this study, PIG3-specific antibodies were used to analyze cellular PIG3 protein levels under control and genotoxic stress conditions. PIG3 protein was localized to the cytoplasm and induced in primary, non-transformed, and transformed cell cultures after exposure to genotoxic agents. The induction of PIG3 was p53-dependent and occurred with delayed kinetics as compared with other p53 downstream targets, such as p21 and MDM2. Using a p53-inducible cell model system, in which p53-mediated growth arrest is reversible, we found that PIG3 levels were increased during p53-mediated growth arrest. Interestingly, elevated levels of PIG3 were maintained in cells that resumed cycling in the absence of ectopic p53 expression, suggesting that PIG3 is a long-lived reporter, which may be useful for detecting transient activation of p53.

Chimera analysis of troponin I domains that influence Ca(2+)-activated myofilament tension in adult cardiac myocytes.

The goal of this study was to investigate isoform-specific functional domains of the inhibitory troponin subunit, troponin I (TnI), as it functions within the intact myofilaments of adult cardiac myocytes. Adenovirus-mediated gene transfer was used to deliver and express a TnI chimera composed of the amino terminus of cardiac TnI (cTnI) and the carboxy terminus of slow skeletal TnI (ssTnI) in adult rat cardiac myocytes. The TnI chimera, designated N-card/slow-C TnI, was expressed and incorporated into myofilaments after gene transfer, without detectable changes in contractile protein stoichiometry or sarcomere architecture. Interestingly, force at submaximal Ca(2+) levels was markedly elevated in single permeabilized myocytes expressing the N-card/slow-C TnI chimera relative to force generated in adult myocytes expressing ssTnI or cTnI. Based on these results, a hierarchy of myofilament Ca(2+) sensitivity is emerging by use of TnI chimera analysis, with the order of sensitivity being N-card/slow-C TnI>>ssTnI>>cTnI. These results also strongly suggest that independent isoform-specific domains in both the amino and carboxy portions of TnI influence myofilament Ca(2+) sensitivity. In additional studies carried out under pathophysiological ionic conditions (pH 6.2), the dramatic acidosis-induced decrease in myofilament Ca(2+) sensitivity observed in myocytes expressing cTnI was blunted in myocytes expressing N-card/slow-C TnI in a manner similar to that in ssTnI-expressing myocytes. These results demonstrate that there is a pH-sensitive domain residing in the carboxy-terminal portion of TnI. The dissection of isoform-specific functional domains under physiological and acidic pH conditions demonstrates the utility of TnI chimeras for analysis of TnI function and provides important insights into the overall function of TnI within the intact myofilament of adult cardiac myocytes.

Functional analysis of troponin I regulatory domains in the intact myofilament of adult single cardiac myocytes.

Troponin I is the putative molecular switch for Ca(2+)-activated contraction within the myofilament of striated muscles. To gain insight into functional troponin I domain(s) in the context of the intact myofilament, adenovirus-mediated gene transfer was used to replace endogenous cardiac troponin I within the myofilaments of adult cardiac myocytes with the slow skeletal isoform or a chimera of the slow skeletal and cardiac isoforms. Efficient expression and myofilament incorporation were observed in myocytes with each exogenous troponin I protein without detected changes in the stoichiometry of other contractile proteins and/or sarcomere architecture. Contractile function studies in single, permeabilized myocytes expressing exogenous troponin I provided support for the presence of a Ca(2+)-sensitive regulatory domain in the carboxyl terminus of troponin I and a second, newly defined Ca(2+)-sensitive domain residing in the amino terminus of troponin I. Additional experiments demonstrated that the isoform-specific, acidic pH-induced contractile dysfunction in myocytes appears to lie in the carboxyl terminus of troponin I. Functional results obtained from adult cardiac myocytes expressing the chimera or isoforms of troponin I now define multiple troponin I regulatory domains operating in the intact myofilament and provide new insight into the Ca(2+)-sensitive properties of troponin I during contraction.

Effects of myosin heavy chain isoform switching on Ca2+-activated tension development in single adult cardiac myocytes.

Cardiac myosin heavy chain (MHC) isoforms are known to play a key role in defining the dynamic contractile behavior of the heart during development. It remains unclear, however, whether cardiac MHC isoforms influence other important features of cardiac contractility, including the Ca2+ sensitivity of isometric tension development. To address this question, adult rats were treated chemically to induce the hypothyroid state and cause a transition in the ventricular cardiac MHC isoform expression pattern from predominantly the alpha-MHC isoform to exclusively the beta-MHC isoform. We found a significant desensitization in the Ca2+ sensitivity of tension development in beta-MHC-expressing ventricular myocytes (pCa50=5. 51+/-0.03, where pCa is -log[Ca2+], and pCa50 is pCa at which tension is one-half maximal) compared with that in predominantly alpha-MHC-expressing myocytes (pCa50=5.68+/-0.05). No differences between the 2 groups were observed in the steepness of the tension-pCa relationship or in the maximum isometric force generated. Instantaneous stiffness measurements were made that provide a relative measure of changes in the numbers of myosin crossbridges attached to actin during Ca2+ activation. Results showed that the relative stiffness-pCa relationship was shifted to the right in beta-MHC-expressing myocytes compared with the alpha-MHC-expressing cardiac myocytes (pCa50=5.47+/-0.05 versus 5.76+/-0.05, respectively). We conclude that MHC isoform switching in adult cardiac myocytes alters the Ca2+ sensitivity of stiffness and tension development. These results suggest that the activation properties of the thin filament are in part MHC isoform dependent in cardiac muscle, indicating an additional role for MHC isoforms in defining cardiac contractile function.

Stability of the contractile assembly and Ca2+-activated tension in adenovirus infected adult cardiac myocytes.

Adenovirus-mediated gene transfer into adult cardiac myocytes in primary culture is a potentially useful method to study the structure and function of the contractile apparatus. However, the consequences of adenovirus infection on the highly differentiated state of the cultured myocyte have not been determined. We report here a detailed analysis of myofilament structure and function over time in primary culture and after adenovirus infection. Adult rat ventricular myocytes in primary culture were infected with a recombinant adenovirus vector expressing either the LacZ or alkaline phosphatase reporter gene. Control and infected myocytes were collected at days 0-7 post-isolation/infection, and myofilament isoform expression was determined by SDS-PAGE and Western blot. Laser scanning densitometry showed that the alpha- to beta-myosin heavy chain ratio, the stoichiometry of the myosin light chains and the expression of the adult troponin T isoform did not change over time in culture or with adenovirus treatment. Importantly, examination of Ca2+-activated tension in single myocytes showed no change in the shape or position of the tension-pCa relationship in the control and adenovirus infected myocytes during primary culture. These results indicate that the structure and function of adult cardiac myocytes are stable in short term primary culture and are not affected by adenovirus infection per se, and therefore provide the foundation for the use of adenovirus-mediated myofilament gene transfer to study contractile apparatus structure and function in adult cardiac myocytes.

Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function.

The functional significance of the developmental transition from slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) isoform expression in cardiac myocytes remains unclear. We show here the effects of adenovirus-mediated ssTnI gene transfer on myofilament structure and function in adult cardiac myocytes in primary culture. Gene transfer resulted in the rapid, uniform, and nearly complete replacement of endogenous cTnI with the ssTnI isoform with no detected changes in sarcomeric ultrastructure, or in the isoforms and stoichiometry of other myofilament proteins compared with control myocytes over 7 days in primary culture. In functional studies on permeabilized single cardiac myocytes, the threshold for Ca2+-activated contraction was significantly lowered in adult cardiac myocytes expressing ssTnI relative to control values. The tension-Ca2+ relationship was unchanged from controls in primary cultures of cardiac myocytes treated with adenovirus containing the adult cardiac troponin T (TnT) or cTnI cDNAs. These results indicate that changes in Ca2+ activation of tension in ssTnI-expressing cardiac myocytes were isoform-specific, and not due to nonspecific functional changes resulting from overexpression of a myofilament protein. Further, Ca2+-activated tension development was enhanced in cardiac myocytes expressing ssTnI compared with control values under conditions mimicking the acidosis found during myocardial ischemia. These results show that ssTnI enhances contractile sensitivity to Ca2+ activation under physiological and acidic pH conditions in adult rat cardiac myocytes, and demonstrate the utility of adenovirus vectors for rapid and efficient genetic modification of the cardiac myofilament for structure/function studies in cardiac myocytes.

Gene transfer into mouse embryonic stem cell-derived cardiac myocytes mediated by recombinant adenovirus.

The main purpose of this study was to examine, for the first time, the ability of recombinant adenovirus to mediate gene transfer into cardiac myocytes derived from mouse embryonic stem (ES) cells differentiating in vitro. In addition, observations were made on the effect of adenovirus infection on cardiac myocyte differentiation and contractility in this in vitro system of cardiogenesis. ES cell cultures were infected at various times of differentiation with a recombinant adenovirus vector (AdCMVlacZ) containing the bacterial lacZ gene under the control of the cytomegalovirus (CMV) promoter. Expression of the lacZ reporter gene was determined by histochemical staining for beta-galactosidase activity. LacZ expression was not detected in undifferentiated ES cells infected with AdCMVlacZ. In contrast, infection of differentiating ES cell cultures showed increasing transgene expression with continued time in culture. Expression in ES-cell-derived cardiac myocytes was demonstrated by codetection of beta-galactosidase activity and troponin T with indirect immunofluorescence. At 24 h postinfection, approximately 27% of the cardiac myocytes were beta-galactosidase positive, and lacZ gene expression appeared to be stable for up to 21 d postinfection. Adenovirus infection had no apparent effect on the onset, extent, or duration of spontaneously contracting ES-cell-derived cardiomyocytes, indicating that cardiac differentiation and contractile function were not significantly altered in the infected cultures. The demonstration of adenovirus-mediated gene transfer into ES-cell-derived cardiac myocytes will aid studies of gene expression with this in vitro model of cardiogenesis and may facilitate future studies involving the use of these myocytes for grafting experiments in vivo.

Ultrastructure and cell-cell coupling of cardiac myocytes differentiating in embryonic stem cell cultures.

Differentiation cultures of embryonic stem (ES) cells can be a useful in vitro system for understanding cardiac myocyte development. However, cell morphometry, sarcomere development, and functional cell-cell junction formation have not been examined in detail to determine whether ES cell-derived cardiac myocytes exhibit structural and functional characteristics similar to cardiac myocytes within the developing heart. Therefore, we examined cellular dimensions, sarcomere formation, and cell-cell contacts in differentiating cardiac myocytes derived from mouse D3-ES cell cultures. Cells exhibited rod-shaped morphology and had single centrally located nuclei, typical of maturing cardiac myocytes. The cellular dimensions of 59 individual cardiac myocytes within contracting foci of ES cell cultures were analyzed (length = 42.2 +/- 2.1 microns, area = 197 +/- 19 microns2, and diameter = 5.5 +/- 0.3 microns) and found to be similar to myocytes in vivo. Transmission electron micrographs of ES cell-derived cardiac myocytes indicated myofibrillar architecture ranged from sparse and disorganized to densely packed, parallel arrays of myofibrils organized into mature sarcomeres. This pattern of myofibrillar assembly in maturing sarcomeres was similar to that observed during in vivo myocyte differentiation. Another hallmark of cardiac development is the formation of intercalated discs, which functionally couple adjacent cardiac myocytes. Electron micrographs indicated nascent intercalated discs were forming in foci of ES cell-derived cardiac myocytes. In addition, indirect immunostaining with anti-connexin 43 antibody (Ab), a monoclonal Ab to the gap junction component of the intercalated disc, indicated that gap junctions were present in contracting ES cell foci. Furthermore, microinjection of single cardiac myocytes with Lucifer yellow (2.5 microM) resulted in the spread of fluorescence to adjacent cells within a contracting focus, an indication of functional cell-cell coupling across these gap junctions. Together, these results indicate ES cell-derived cardiac myocytes exhibit cell morphology, sarcomere formation, and cell-cell junctions similar to those observed in cardiac myocytes developing in vivo.

Embryonic stem cell cardiogenesis applications for cardiovascular research.

Mouse embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the preimplantation blastocyst. These cells can be maintained in culture in an undifferentiated state, or they can be induced to differentiate in vitro into multiple cell types, including spontaneously beating cardiac myocytes. The ability to engineer these ES cells genetically, together with their noted rapid differentiation into cardiac myocytes in vitro, makes this a useful tool for the study of cardiac gene expression and function. This in vitro cardiogenesis system may be particularly advantageous for pharmacological studies focusing on discovery of cardioactive drugs and for specifying the functional alterations associated with ablated or mutated cardiac genes that result in a lethal phenotype in vivo. (Trends Cardiovasc Med 1997;7:63-68). © 1997, Elsevier Science Inc.

Suspected bentonite toxicosis in a cat from ingestion of clay cat litter.

A 2 1/2-y-old spayed female cat was presented for lethargy and weakness. The cat was hypokalemic (3.1 m Eq K/L) and severely anemic (60% PVC, 1.3 g hemoglobin/dL). The cat was known to ingest bentonite-containing cat litter. It recovered with treatment of i.v. fluids, electrolytes and whole blood transfusion and was discharged. Two months later the cat was presented again with signs similar to those seen previously. This occurred 1 mo after the owner resumed the use of bentonite-containing cat litter. The signs were remarkably similar to those reported in humans from the chronic ingestion of bentonite clays. Bentonite toxicosis is suggested by the coexistence of hypokalemia hypochromic anemia in cats presented with lethargy and muscle weakness.

Troponin I isoform expression is developmentally regulated in differentiating embryonic stem cell-derived cardiac myocytes.

We studied troponin I (TnI) isoform expression in the mouse embryonic stem (ES) cell model of cardiogenesis as an essential first step to understanding the relationship between TnI isoform transitions and myofibrillar function. Cultures of differentiating ES cells were grown on coverslips to permit microscopic inspection of foci of spontaneously contracting cardiac myocytes developing in culture. TnI expression was followed over time to test whether the cardiac myocytes undergo the developmental pattern of expression characteristic of vertebrate cardiogenesis, in which slow skeletal TnI (ssTnI) is expressed initially, followed by induction of cardiac (cTnI) isoform expression. Cardiac TnI expression was examined using the cardiac-specific, monoclonal TI-1 antibody (Ab) while all striated muscle ThI isoforms were detected using the monoclonal TI-4 Ab. Cardiac-specific TnI expression was detected in only 8% (8/96) of foci contracting less than 5 days while TI-4 positive staining was present in 95% (71/73) of foci. These results indicate that other striated muscle TnI isoforms were being expressed in most of the TI-4 positive staining foci. The proportion of contracting foci expressing the cardiac isoform increased steadily over time, such that 100% of foci contracting more than 20 days (13/13) stained positive with the TI-1 Ab. Dual labeling experiments with both TI-1 and TI-4 anti-TnI Abs in the same culture confirmed that within each foci, the area expressing cTnI increased with the days of spontaneous contraction. Western blot analysis of micro-dissected ES cell derived cardiac myocytes confirmed that TI-4 immunostaining at early developmental time points represented ssTnI, and not the fast skeletal TnI isoform. We conclude that ES cell-derived cardiac myocytes display the developmental induction of cardiac TnI expression characteristic of vertebrate cardiac development. Thus, this model should be useful for studying the regulation and functional significance of TnI isoform expression during in vitro cardiogenesis.

CGP-48506 increases contractility of ventricular myocytes and myofilaments by effects on actin-myosin reaction.

We measured the effects of the benzodiazocine derivative, CGP-48506 (5-methyl-6-phenyl-1,3,5,6-tetrahydro-3,6-methano-1, 5-benzodiazocine-2,4-dione), on contraction of intact myocytes and permeabilized fibers of rat ventricular muscle. CGP-48506 is unique in that it is able to sensitize cardiac myofilaments to Ca2+, but unlike all other agents in this class, it is not an inhibitor of type III phosphodiesterase. When added to isolated intact myocytes, CGP-48506 significantly increased the amplitude of cell shortening with little or no change in the Ca2+ transient, as determined by the fluorescence ratio of fura 2. The late phase of the relation between fura 2 ratio and cell length was shifted to the left in the presence of CGP-48506. CGP-48506 also induced a relatively small decrease in diastolic length. However, compared with the thiadiazinone EMD-57033, CGP-48506 had a much smaller effect on diastolic length at concentrations in which there was a bigger inotropic effect. When added to solutions bathing detergent-extracted (skinned) fiber bundles, CGP-48506 increased maximum force. CGP-48506 also increased submaximal force and shifted the pGa-force relation to the left. However, compared with EMD-57033, there was less of an effect of CGP-48506 on force at relatively high pCa values. CGP-48506 did not alter Ca2+ binding to myofilament troponin C. CGP-48506 was able to reverse inhibition of contraction induced by butanedione monoxime both in intact cells and in skinned fiber bundles. Our results indicate that CGP-48506, like EMD-57033, is a positive inotropic agent working through a direct effect downstream from troponin C. CGP-48506, however, appears to have a unique mechanism resulting in less effect on diastolic function.

Altered Ca2+ sensitivity of tension in single skeletal muscle fibres from MyoD gene-inactivated mice.

1. Single, fast glycolytic skeletal muscle fibres were isolated from wild-type (MyoD+/+) and MyoD mutant mice (MyoD-/-), which lack a functional copy of the MyoD gene. Fibres were chemically permeabilized to permit manipulation and control of the ionic environment of the otherwise intact myofilament apparatus. 2. Results show a fivefold greater variability in the [Ca2+] required for half-maximum tension generation among individual MyoD-/- fibres in comparison with controls (p < 0.05). 3. Consistent with this finding, Western blot analysis showed a sevenfold greater variability in the isoform expression pattern of the thin filament regulatory protein troponin T in Myod-/- compared with control fibres (p < 0.05). 4. Electrophoretic analysis of single-fibre segments indicated no apparent alteration in the isoform expression pattern of other regulatory and contractile proteins. In addition, other parameters of contractile function, including velocity of unloaded shortening, and maximum force production, were not significantly different between MyoD-/- and MyoD ø fibres. 5. These findings indicate that the thin filament structure- function relationship is altered due to the MyoD mutation and suggest that MyoD plays a role in establishing and/or maintaining the differentiated phenotype of adult fast skeletal muscle fibres.