Myofibril Relaxation Research Articles

PKA Phosphorylation of Cardiac Troponin I Modulates Activation and Relaxation Kinetics of Ventricular Myofibrils

Protein kinase A (PKA) phosphorylation of myofibril proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility and relaxation. PKA targets the N-terminus (Ser-23/24) of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. The effect of PKA-mediated phosphorylation on the magnitude of contraction has been studied in some detail, but little is known about how it modulates the kinetics of thin filament activation and myofibril relaxation as Ca2+ levels vary. Troponin C (cTnC) interaction with cTnI (C-I interaction) is a critical step in contractile activation that can be modulated by cTnI phosphorylation. We tested the hypothesis that altering C-I interactions by PKA, or by cTnI phosphomimetic mutations (S23D/S24D-cTnI), directly affects thin filament activation and myofilament relaxation kinetics. Rat ventricular myofibrils were isolated and endogenous cTn was exchanged with either wild-type cTnI, or S23D/S24D-cTnI recombinant cTn. Contractile mechanics were monitored at maximum and submaximal Ca2+ concentrations. PKA treatment of wild-type cTn or exchange of cTn containing S23D/S24D-cTnI resulted in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow). These effects were greater for submaximal Ca2+ activated contractions. PKA treatment also reduced the rate of contractile activation (kACT) at maximal, but not submaximal Ca2+, and reduced the Ca2+ sensitivity of contraction. Using a fluorescent probe coupled to cTnC (C35S-IANBD), the Ca2+-cTn binding affinity and C-I interaction were monitored. Ca2+ binding to cTn (pCa50) was significantly decreased when cTnI was phosphorylated by PKA (ΔpCa50 = 0.31). PKA phosphorylation of cTnI also weakened C-I interaction in the presence of Ca2+. These data suggest that weakened C-I interaction, via PKA phosphorylation of cTnI, may slow thin filament activation and result in increased myofilament relaxation kinetics, the latter of which could enhance early phase diastolic relaxation during β-adrenergic stimulation.

Abstract 35: 2-deoxy Adenosine Triphosphate Improves Contraction In Human End-stage Heart Failure

We are developing a novel approach to treat heart failure that involves adeno-associated virus vectors to elevate intracellular 2 deoxy-ATP (dATP) via increased expression of the enzyme Ribonucleotide Reductase. Our studies in rodents have shown that substitution of dATP for ATP as the energy substrate increases contraction in striated muscle. Here we report for the first time the effect of dATP on human cardiac muscle contraction. We measured the contractile properties of demembranated multicellular ventricular wall preparations and isolated myofibrils from adult human heart tissue obtained from twelve patients undergoing surgery for cardiac transplantation or placement of left ventricular-assist device for end-stage heart failure. Isometric force at saturating calcium concentration was increased by about ten percent from 38.6±3.8 mN/mm2 to 42.8±4.2 mN/mm2 when dATP was substituted for ATP (P<0.001). The effect was even greater at physiologic calcium concentrations with an approximate increase of thirty percent. Isometric force increased from 21.2 ± 6.1 mN/mm2 to 26.4± 6.4 mN/mm2 (p<0.001) at pCa=5.6 and from 22.6 ± 5.9 mN/mm2 to 27.5± 6.3 mN/mm2 (p<0.001) at pCa=5.8 with dATP for ATP substitution. The result was an increase in the Ca2+ sensitivity of force as the [Ca2+] required to elicit half maximum force (pCa50) increased by 0.08 units from 5.68±0.03 to 5.79±0.03 (N=24, P< 0.001). The maximum rate of force redevelopment (ktr) in demembranated wall muscle increased (0.82±0.01 s-1 vs. 0.62±0.01 s-1, P< 0.05), as was the rate of contractile activation in isolated myofibrils (0.80±0.06 s-1 vs. 0.57±0.06 s-1, P<0.01) suggesting dATP may increase dP/dT in failing human myocardium. Importantly, there was no slowing of relaxation, as the time to 50% and 90% myofibril relaxation were unchanged. Purified myosin from failing human myocardium showed enhanced NTPase activity with dATP (0.89±0.17 s-1/head) vs. ATP (0.55±0.20 s-1/head, P<0.05). In conclusion, the data strongly suggest dATP increases cross-bridge cycling, compared with ATP in failing human myocardium and shows promise in restoring cardiac pump function. These data support a novel myofilament approach for treating heart failure that warrants further pre-clinical evaluation.

The Mechanism of HCM-Related Mutation R21C on the Modulation of C-I Interactions and Contractile Kinetics

The R21C mutation in cTnI, associated with hypertrophic cardiomyopathy (HCM), is the only known mutation within N-terminus of cTnI. The mechanisms by which this mutation leads to heart failure, and how this mutation affects PKA regulation of cTn function and myofibril relaxation still remain elusive. Currently, we are studying this mutation using an integrated experimental-computational approach. We generated triple-mutated cTnI (cTnI-R21C/S2324D) to mimic the phosphorylation. From steady-state fluorescence measurement, which using a fluorescent probe (IANBD) coupled to cTnCC35S, C-I binding affinity (KC-I) and calcium binding affinity (KCa) to whole cTn were monitored. cTnIR21C increased both KC-I and KCa compared to cTnIWT. Phosphorylation of cTnI reduced KCa for both complexes. However, while PKA treatment weakened KC-I for cTnIWT, this effect was not absent in cTnIR21C. To study myofibril contraction/relaxation mechanics and kinetics, endogenous cTn was exchanged with either WT or R21C recombinant cTn, and mechanics were monitored at maximum and submaximal Ca2+ conditions. The kinetics of activation (KACT) is slower at submaximal vs maximal Ca2+ conditions, and not affected by PKA treatment of myofibrils containing either cTnIWT or cTnIR21C. However, PKA treatment of cTn with cTnIWT significantly affected slow phase relaxation, increasing slow phase rate (KREL,slow) and decreasing slow phase duration (tREL, slow), and these effects are more clear at submaximal calcium levels. Interestingly, these PKA mediated effects were blunted in cTnIR21C. Molecular dynamics (MD) simulations of whole cTn with R21C and R21C/S23DS24D substitution are ongoing. Interactions between cNTnC-cNTnI, cNTnC-switch peptide of cTnI, and cTnC-inhibitory peptide of cTnI will be monitored, and calcium binding sites will be studied. Computational results will provide the structural basis at molecular level of how R21C influence C-I binding and relaxation of cardiac muscles. HL65497, HL11197 (MR), 8P41GM103426 (AM & AM).

Cypher/ZASP Is a Novel A-kinase Anchoring Protein

PKA signaling is important for the post-translational modification of proteins, especially those in cardiomyocytes involved in cardiac excitation-contraction coupling. PKA activity is spatially and temporally regulated through compartmentalization by protein kinase A anchoring proteins. Cypher/ZASP, a member of PDZ-LIM domain protein family, is a cytoskeletal protein that forms multiprotein complexes at sarcomeric Z-lines. It has been demonstrated that Cypher/ZASP plays a pivotal structural role in the structural integrity of sarcomeres, and several of its mutations are associated with myopathies including dilated cardiomyopathy. Here we show that Cypher/ZASP, interacting specifically with the type II regulatory subunit RIIα of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. In addition, we show that Cypher/ZASP itself was phosphorylated at Ser(265) and Ser(296) by PKA. Furthermore, the PDZ domain of Cypher/ZASP interacted with the L-type calcium channel through its C-terminal PDZ binding motif. Expression of Cypher/ZASP facilitated PKA-mediated phosphorylation of the L-type calcium channel in vitro. Additionally, the phosphorylation of the L-type calcium channel at Ser(1928) induced by isoproterenol was impaired in neonatal Cypher/ZASP-null cardiomyocytes. Moreover, Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel. Taken together, our data strongly suggest that Cypher/ZASP not only plays a structural role for the sarcomeric integrity, but is also an important sarcomeric signaling scaffold in regulating the phosphorylation of channels or contractile proteins.

The HCM-Associated Cardiac Troponin T Mutation K280N Increases the Energetic Cost of Tension Generation in Human Cardiac Myofibrils

A novel homozygous mutation in the TNNT2 gene encoding cardiac troponin T (cTnT K280N) was identified in one HCM patient undergoing cardiac transplantation. mRNA and Mass Spectrometry analyses revealed expression of the mutant alleles without evidence of haploinsufficiency. Kinetics of contraction and relaxation of myofibrils from a frozen left ventricular sample of the K280N HCM patient were compared to those of “control” myofibrils (from donor hearts, from aortic stenosis patients, and from HCM patients negative for sarcomeric protein mutations). Preparations, mounted in a force recording apparatus (15 °C), were maximally Ca2+-activated (pCa 4.5) and fully relaxed (pCa 9) by rapid (<10 ms) solution switching. The rate constant of active tension generation following maximal Ca2+ activation (kACT) was markedly faster in K280N myofibrils (ca. 1.7 s−1) compared to controls (0.7-1 s−1). The rate constant of isometric relaxation (slow kREL) was 2-3-fold faster in K280N myofibrils than in controls, evidence that the apparent rate with which cross-bridges leave the force generating states is accelerated in the mutant preparations. The results suggest that the energy cost of tension generation is increased in the K280N sarcomeres. Simultaneous measurements of maximal isometric ATPase and Ca2+-activated force in Triton-permeabilized left ventricular muscle strips from the K280N sample demonstrated that tension cost was much higher in the K280N than in control myocardium. Replacement of the mutant protein by exchange with wild-type recombinant human Tn in the K280N myofibrils reduced both kACT and slow kREL close to control values. This indicates that the HCM-associated TNNT2 mutation K280N primarily alters apparent cross-bridge kinetics and impairs sarcomere energetics. Supported by the 7th Framework Program of the European Union, “BIG-HEART” grant agreement 241577.

Mechanisms of Altered Cardiac Myofibril Relaxation Caused by the Cardiac Troponin-I R145G Mutation

The cardiac troponin I (cTnI) mutation R145G, associated with hypertrophic cardiomyopathy, increases the calcium sensitivity of contraction and alters the kinetics of relaxation. It is unclear whether this results from altered calcium binding of cTn, altered interaction with troponin C (cTnC), or both. It is also unclear how this alters protein kinase A (PKA) mediation of cTn function and myofibril relaxation. We are studying this using an experimental and computational approach. Steady-state fluorescence experiments indicate a significant increase in calcium binding affinity of cTn with R145G vs. wild-type (WT) cTnI. PKA phosphorylation of cTnI resulted in a similar reduction in this affinity for both cTn complexes. Interaction of cTnC with cTnI (C-I) was greater with R145G vs. WT-cTnI. PKA treatment of cTnI weakened C-I interactions for WT, but not R145G-cTnI. PKA treatment of WT-cTnI exchanged rat ventricular myofibrils significantly affected the slow phase of relaxation, increasing the slow phase rate (kREL,slow) and decreasing slow phase duration (tREL,slow). These effects were larger at submaximal calcium levels. Interestingly, R145G-cTnI blunted PKA mediated effects. Molecular dynamics simulations of the crystal structure of cTn were used to investigate the molecular contacts between the cTnI switch peptide region and cNTnC for WT and R145G-cTnI. Preliminary results suggest a profound effect of the mutation on the interaction of residues 147-149 cTnI with cNTnC. For R145G cTn, switch peptide residues 148 and 149 lose most of their strong contacts with cNTnC, and residue 147 forms strong contacts not observed with WT-cTnI. The results suggest the C-I interaction as a molecular level site of R145G effects on relaxation of cardiac muscle. HL65497 (MR), AHA 7400069 (VR), GM103426-19S1 (PA), AHA 12POST11570005 (SL), GM31749 (JAM), NSF MCB 1020765 (JAM), HHMI (JAM), SDSC (JAM), CTBP (JAM).

Altered Cross Bridge Kinetics in Skeletal Myofibrils from NEBΔex55, a Novel Mouse Model of Nebulin-Based Nemaline Myopathy

Nemaline Myopathy is the most common non-dystrophic congenital myopathy, clinically characterized by muscle weakness. The disease is associated with mutations in the nebulin gene and the nebulin-based disease is referred to as NEM2. Recent work on skinned muscle fibres from NEM2 patients revealed remarkable phenotypic similarities to fibres from nebulin KO mice (Ottenheijm et al, 2012). Here we investigated mechanics and kinetics of single myofibrils from a novel NEM2 mouse model (NEB Δex55) that mimics a deletion in the nebulin gene found in a large group of NEM2 patients. We used rapid solution switching (Tesi et al.,2002) to compare maximal tension and kinetics of contraction and relaxation of myofibrils isolated from frozen skeletal muscles (tibialis cranialis of neonatal mice) of WT and NEB Δex55 mice. Myofibrils, mounted in a force recording apparatus (15 °C), were maximally Ca2+-activated (pCa 4.5) and fully relaxed (pCa 9.0). Maximal isometric tension was markedly reduced in NEB Δex55 mouse myofibrils (49.7±10.6 mN mm−2n=11) compared to WT (135.3±16.9mN mm−2n=9).The rate constant of active tension generation following maximal Ca2+ activation (kACT) was significantly reduced inNEB Δex55 mouse myofibrils (1.46±0.07s−1) compared to WT (2.75±0.27 s−1). Force relaxation kinetics was remarkably faster in NEB Δex55 mouse myofibrils than in WT, evidence that the apparent rate with which cross-bridges leave the force generating states is accelerated in the NEB Δex55sarcomeres. Reduction of the rate with which cross-bridges enter force generating states and of cross bridge dissociation can markedly contribute to reducing maximal tension. This is expected to increase the energetic cost of tension generation of the NEB Δex55sarcomeres. Results suggest that nebulin plays a significant role in contraction regulation and that altered cross bridge kinetics contribute to NEM2 pathogenesis

Protein kinase A changes calcium sensitivity but not crossbridge kinetics in human cardiac myofibrils

We investigated the effect of PKA treatment (1 U/ml) on the mechanical properties of isolated human cardiac myofibrils. PKA treatment was associated with significant incorporation of radiolabeled phosphate into several sarcomeric proteins including troponin I and myosin binding protein C and was also associated with a right shift in the tension-pCa relation (ΔpCa(50) = 0.2 ± 0.1). PKA treatment also caused right shifts in the pCa dependence of the rate of tension development, tension redevelopment, and the linear and exponential phases of myofibril relaxation. However, there was no change in the same measures of crossbridge turnover when expressed as a function of tension. We conclude that the changes in crossbridge kinetics as a function of calcium concentration reflect a reduced tension due to a lower calcium sensitivity and that the relationship between crossbridge kinetics and tension was unchanged, indicating no direct effect of PKA treatment on crossbridge cycling.

The HCM-Associated Cardiac Troponin T Mutation K273N Accelerates Tension Generation and Relaxation in Human Cardiac Myofibrils

In spite of extensive work on the functional sequelae of Hypertrophic CardioMyopathy (HCM)-associated mutations in sarcomeric proteins, the mechanisms by which the mutant proteins cause the disease have not been definitely identified. Here we use the single myofibril technique (Tesi et al., Biophys. J., 2002, 83, 2142-2151) to compare the kinetics of contraction and relaxation of myofibrils isolated from frozen left ventricular samples of one homozygous HCM patient carrying the cardiac Troponin T (cTnT) mutation K273N (underwent heart transplantation) to those from “control” hearts. Preparations, mounted in a force recording apparatus (15 °C), were maximally Ca2+-activated (pCa 4.5) and fully relaxed (pCa 9) by rapid (<10 ms) solution switching. The rate constant of active tension generation following maximal Ca2+ activation (kACT) was markedly faster in the myofibrils from the K273N patient (1.7- 2 s−1) compared to controls (0.7-1 s−1). Replacement of the mutant protein by exchange with wild-type recombinant human cTn reduced kACT of HCM myofibrils close to control values (1 s−1). Force relaxation kinetics upon Ca2+ removal were also faster in K273N myofibrils than in controls, evidence that the apparent rate with which cross-bridges leave the force generating states is accelerated in the HCM preparations. The results indicate that the HCM-associated cTnT mutation K273N alters apparent cross-bridge kinetics. This can lead to increased energy cost of tension generation that may be central to the HCM disease process. Supported by the 7th Framework Program of the European Union, “BIG-HEART” grant agreement 241577, & Telethon-Italy GGP07133.

Desensitizing Effect of N-terminal Truncated cTnI In RCM Myofibrils

Cardiac TnI (cTnI) mutations have been associated with the development of restrictive cardiomyopathy (RCM) characterized by a Ca2+ hypersensitivity and diastolic dysfunction in cardiac myofibrils. Whereas cTnI N-terminal deletion (cTnI-ND) caused by restricted proteolysis in cardiac adaptation to stress manifests a lower left ventricular end diastolic pressure and an enhanced ventricular diastolic function. By crossing the RCM cTnI R193H transgenic mice (cTnI193His) with cTnI-ND transgenic mice (cTnI-ND) that contain 100% cTnI-ND in the heart, we have obtained double TG mice containing both the cTnI R193H mutant and cTnI-ND. In this study, by using these TG mouse lines, we have investigated the desensitizing effect of cTnI-ND on the RCM cTnI mutant mice and myofibrils. Our survival data for these mice indicated that cTnI-ND greatly reduced the mortality of the RCM cTnI193His mice. Ca2+ sensitivity measured in skinned myofibrils confirmed that increased myofibril Ca2+ sensitivity was the major mechanism that resulted in impaired relaxation and diastolic dysfunction in RCM cTnI193His mice and that cTnI-ND could reverse the cellular dysfunction by desensitizing the myofibrils to Ca2+. The PKA stimulation assays showed that cTnI193His myofibrils were able to respond to PKA activation, resulting in a right-shift of pCa curve after PKA treatment. However, since the myofibrils from cTnI-ND hearts lacked Ser residues 23 and 24, they had no response to PKA stimulation, showing a similar pCa curve before or after PKA. Our data have, for the first time, demonstrated a desensitizing effect by an endogenous myofibril protein proteolysis without the intervention of β-adrenergic stimulation mediated cTnI phosphorylation. The desensitizing function in cTnI-ND hearts indicates that the removal of cTnI N-terminal extension by restricted proteolysis represents a novel mechanism to improve myofibril relaxation and cardiac diastolic function in cardiac adaptation to hemodynamic and inotropic stresses.

Mechanism of cross-bridge detachment in isometric force relaxation of skeletal and cardiac myofibrils.

Skeletal and cardiac muscle relaxation is governed by the interplay between two macromolecular systems: (i) membrane bound Ca2+ transport proteins and (ii) sarcomeric proteins. Photolysis experiments in skinned muscle preparations and fast solution switching studies in single myofibrils offer means for isolating sarcomeric mechanisms of relaxation from those related to myoplasmic Ca2+ removal. Single myofibril experiments have recently shown that cross-bridge mechanics and detachment kinetics are the major determinants of the time course of relaxation. Full force decay in myofibrils occurs in two phases: a slow one followed by a rapid one. The latter is initiated by sarcomere 'give' and dominated by inter-sarcomere dynamics while the former occurs under nearly isometric conditions. Strong evidence has been found that the slow rate of force decay in myofibril relaxation reflects the rate at which cross-bridges leave force-generating states under isometric conditions. Dissection of chemo-mechanical transduction process in myofibrils indicates that both forward and backward transitions of cross-bridges from force-generating to non-force-generating states contribute to muscle relaxation.

Phosphorylation of troponin I by protein kinase A accelerates relaxation and crossbridge cycle kinetics in mouse ventricular muscle.

Phosphorylation of cardiac myofibrils by cAMP-dependent protein kinase (PKA) can increase the intrinsic rate of myofibrillar relaxation, which may contribute to the shortening of the cardiac twitch during beta-adrenoceptor stimulation. However, it is not known whether the acceleration of myofibrillar relaxation is due to phosphorylation of troponin I (TnI) or of myosin binding protein-C (MyBP-C). To distinguish between these possibilities, we used transgenic mice that overexpress the nonphosphorylatable, slow skeletal isoform of TnI in the myocardium and do not express the normal, phosphorylatable cardiac TNI: The intrinsic rate of relaxation of myofibrils from wild-type and transgenic mice was measured using flash photolysis of diazo-2 to rapidly decrease the [Ca(2+)] within skinned muscles from the mouse ventricles. Incubation with PKA nearly doubled the intrinsic rate of myofibrillar relaxation in muscles from wild-type mice (relaxation half-time fell from approximately 150 to approximately 90 ms at 22 degrees C) but had no effect on the relaxation rate of muscles from the transgenic mice. In parallel studies with intact muscles, we assessed crossbridge kinetics indirectly by determining f(min) (the frequency for minimum dynamic stiffness) during tetanic contractions. Stimulation of beta-adrenoceptors with isoproterenol increased f(min) from 1.9 to 3.1 Hz in muscles from wild-type mice but had no effect on f(min) in muscles from transgenic mice. We conclude that the acceleration of myofibrillar relaxation rate by PKA is due to phosphorylation of TnI, rather than MyBP-C, and that this may be due, at least in part, to faster crossbridge cycle kinetics.

Spontaneous oscillatory contraction of sarcomeres in skeletal myofibrils.

We found that the lengths of all sarcomeres spontaneously oscillated in an isolated skeletal myofibril, when both ends were fixed, submillimolar to millimolar concentrations of ATP, ADP and inorganic phosphate (Pi) were present, and Ca2+ was removed. Narrowing and widening of an H-zone and an I-band were observed corresponding to the shortening and lengthening of a sarcomere, suggesting that thick and thin filaments slide past each other. The oscillation of each sarcomere was asymmetrical, consisting of a rapid lengthening phase and a slow shortening phase. The period of oscillation was about 3s; the peak-to-peak amplitude of oscillation reached as much as 30% of the average sarcomere length. The propagation of the sarcomere oscillation along the long axis of the myofibril was observed occasionally in single myofibrils and frequently in bundles of myofibrils. The 'state'-diagram showing the concentration range of ADP and Pi in which contraction, oscillation or relaxation of myofibrils occurs in the presence of ATP and the absence of Ca2+ suggested that the oscillation is a third state of skeletal muscle located in between the contracting and relaxing states.

Quantitative ultrastructural criteria of myocardial damage during postischemic reperfusion

Swollen clarified mitochondria were found during postischemic reperfusion in cardiomyocytes with and without signs of myofibril relaxation. The values of morphometric parameters of mitochondria were similar in both groups of cells studied. Mean indexes characterizing the condition of cardiomyocyte mitochondria may serve as quantitative criteria of the severity of myocardial damage: surface to volume, ratio, visual index of mitochondrial condition and relative volume density, i.e. volume density with respect to control cell volume.

The effects of raised phosphate level on the energy metabolism, contractile function, and fine structure of oxygenated and oxygen-deficient myocardium.

The possible role of inorganic phosphate (Pi) in depressing the contractility of oxygen-deficient myocardium was investigated by examining the effects of 30 mM Pi on the cardiac function and myocardial biochemistry and fine structure in normoxic and anoxic Langendorff-perfused isolated rat hearts. In normoxia, the intracellular Pi level increased three-fold, the ATP content remained normal, and there was moderate loss of glycogen only. Contractile performance (as assessed from systolic pressure recordings) was significantly depressed, as was the heart rate for the first 10 min. The myocardial fine structure showed persistent glycogen, marked relaxation of myofibrils, and a higher incidence of vacuolation than in hearts with normal Pi. In anoxia, the intracellular Pi level was comparable with that of the perfusate and both ATP and glycogen were severely depleted. Contractile performance and heart beat ceased completely at 15 min, although in anoxic controls both persisted at low levels for at least 25 min. In anoxia, Pi also depressed coronary flow rate. In the inner half of the ventricular wall of oxygen-depleted hearts, where flow became reduced after 15 or more min, Pi markedly reduced the formation of intramitochondrial densities and augmented mitochondrial swelling and ischaemic contracture, which extended out through the mid-myocardium. In the outer half of the wall, where flow remained high, it promoted severe dilatation of the sarcoplasmic reticulum vesicles and undifferentiated regions of the intercalated discs. The observed effects in normoxia are probably attributable at least in part to the lowering of the free Ca2+ concentration of the perfusate by the increased Pi level.(ABSTRACT TRUNCATED AT 250 WORDS)

Early phase of myocardial ischemic injury and infarction

The acute effects of ischemia on myocardial tissue are reviewed in this paper with emphasis on how the events observed in experimental myocardial ischemic injury in dogs relate to the sequential changes occurring in the myocardium of man during the first few hours after the onset of acute infarction. The general effects of ischemia on myocardium include those which are secondary to a diminished local supply of substances such as oxygen and metabolites as well as changes resulting from the impaired diffusion of substances such as lactic acid and electrolytes from the poorly perfused ischemic tissue to the general circulation. Cell death (irreversible injury) first develops after 20 minutes of ischemia in areas of maximum injury in the dog heart and infarcts are not fully developed for 60 to 120 minutes after occlusion. Prior to the development of cell death, the severely ischemic cells are reversibly injured and show a variety of alterations from normal. These changes include depletion of supplies of glycogen and high energy phosphate, increased content of lactic acid and hydrogen, relaxation of myofibrils and failure of contraction. Cells which have just died show the same changes as well as mitochondrial, electrolyte, and nuclear defects. Which, if any, of the preceding changes causes the development of irreversibility in ischemic injury remains to be established. Some data is presented in support of the hypothesis that mitochondrial defects may be critical in the genesis of the irreversible state.

The role of magnesium in the relaxation of myofibrils.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTRole of magnesium in the relaxation of myofibrilsAnnemarie Weber, Ruth Herz, and I. ReissCite this: Biochemistry 1969, 8, 6, 2266–2271Publication Date (Print):June 1, 1969Publication History Published online1 May 2002Published inissue 1 June 1969https://doi.org/10.1021/bi00834a005RIGHTS & PERMISSIONSArticle Views92Altmetric-Citations64LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (673 KB) Get e-Alerts Get e-Alerts

Parallel response of myofibrillar contraction and relaxation to four different nucleoside triphophates.

The Mg chelates of ITP, GTP, and UTP in addition to that of ATP were shown to be capable of causing complete relaxation of myofibrils as indicated by the complete inhibition of syneresis and the reduction of the NTPase activity to that of isolated myosin. For ITP and GTP the required concentrations were about 100 times higher than those for ATP, whereas UTP was maximally effective also in low concentrations (0.2 mM). For all NTP's the concentrations for relaxation were related to those necessary for contraction so that NTP concentrations which gave 80-90% maximal NTPase activity in the presence of Ca caused complete relaxation in the absence of Ca. Thus, these experiments do not support the view that relaxation is caused by the binding of NTP to a special inhibitory site but are quite compatible with the idea that relaxation depends on the extent to which the hydrolytic site is saturated with NTP. The Ca concentration required for contraction depends on the nature of the NTP base and its concentration; it is lower for ITP than for ATP and decreases with decreasing concentrations of ITP.

Relationship between ATPase activity and optical density during contraction and relaxation of myofibrils

Contraction and relaxation of myofibrils, as induced by various ATP concentrations, has been studied by measuring simultaneously changes in optical density and ATPase activity. The optical density of myofibrils is increased by low ATP concentrations and decreased by high ATP concentrations. Changes in optical density and syneresis of myofibrils occur immediately after addition of ATP, and are accompanied by the high initial rate of ATPase activity. The ATPase activity of myofibrils is influenced by the ATP concentrations particularly during the initial period, and to a much lower extent after the occurrence of the physical changes in the myofibrils. Addition of Mg 2+ enhances all ATP effects, both on contraction and relaxation of myofibrils. Addition of Ca 2+ produces a small increase of the optical density but a powerful stimulation of the ATPase activity. Inhibition of SH groups produces relaxation of myofibrils even at low ATP concentrations. It is concluded that only the initial high rate of ATPase is expression of the mechano-chemical coupling process. The effects of Mg 2+ and Ca 2+ on this process are discussed.