Gerrie P. Farman

Titin Isoform Variance and Length Dependence of Activation in Skinned Bovine Cardiac Muscle

We have explored the role of the giant elastic protein titin in the Frank‐Starling mechanism of the heart by measuring the sarcomere length (SL) dependence of activation in skinned cardiac muscles with different titin‐based passive stiffness characteristics. We studied muscle from the bovine left ventricle (BLV), which expresses a high level of a stiff titin isoform, and muscle from the bovine left atrium (BLA), which expresses more compliant titin isoforms. Passive tension was also varied in each muscle type by manipulating the pre‐history of stretch prior to activation. We found that the SL‐dependent increases in Ca2+ sensitivity and maximal Ca2+‐activated tension were markedly more pronounced when titin‐based passive tension was high. Small‐angle X‐ray diffraction experiments revealed that the SL dependence of reduction of interfilament lattice spacing is greater in BLV than in BLA and that the lattice spacing is coupled with titin‐based passive tension. These results support the notion that titin‐based passive tension promotes actomyosin interaction by reducing the lattice spacing. This work indicates that titin may be a factor involved in the Frank‐Starling mechanism of the heart by promoting actomyosin interaction in response to stretch.

Iroquois homeobox gene 3 establishes fast conduction in the cardiac His–Purkinje network

Rapid electrical conduction in the His–Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His–Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction–forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for Irx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart.

Molecular dynamics of cyclically contracting insect flight muscle in vivo

Flight in insects—which constitute the largest group of species in the animal kingdom—is powered by specialized muscles located within the thorax. In most insects each contraction is triggered not by a motor neuron spike but by mechanical stretch imposed by antagonistic muscles. Whereas ‘stretch activation’ and its reciprocal phenomenon ‘shortening deactivation’ are observed to varying extents in all striated muscles, both are particularly prominent in the indirect flight muscles of insects. Here we show changes in thick-filament structure and actin–myosin interactions in living, flying Drosophila with the use of synchrotron small-angle X-ray diffraction. To elicit stable flight behaviour and permit the capture of images at specific phases within the 5-ms wingbeat cycle, we tethered flies within a visual flight simulator. We recorded images of 340 µs duration every 625 µs to create an eight-frame diffraction movie, with each frame reflecting the instantaneous structure of the contractile apparatus. These time-resolved measurements of molecular-level structure provide new insight into the unique ability of insect flight muscle to generate elevated power at high frequency.

Aging Enhances Indirect Flight Muscle Fiber Performance yet Decreases Flight Ability in Drosophila

We investigated the effects of aging on Drosophila melanogaster indirect flight muscle from the whole organism to the actomyosin cross-bridge. Median-aged (49-day-old) flies were flight impaired, had normal myofilament number and packing, barely longer sarcomeres, and slight mitochondrial deterioration compared with young (3-day-old) flies. Old (56-day-old) flies were unable to beat their wings, had deteriorated ultrastructure with severe mitochondrial damage, and their skinned fibers failed to activate with calcium. Small-amplitude sinusoidal length perturbation analysis showed median-aged indirect flight muscle fibers developed greater than twice the isometric force and power output of young fibers, yet cross-bridge kinetics were similar. Large increases in elastic and viscous moduli amplitude under active, passive, and rigor conditions suggest that median-aged fibers become stiffer longitudinally. Small-angle x-ray diffraction indicates that myosin heads move increasingly toward the thin filament with age, accounting for the increased transverse stiffness via cross-bridge formation. We propose that the observed protein composition changes in the connecting filaments, which anchor the thick filaments to the Z-disk, produce compensatory increases in longitudinal stiffness, isometric tension, power and actomyosin interaction in aging indirect flight muscle. We also speculate that a lack of MgATP due to damaged mitochondria accounts for the decreased flight performance.

Cardiac Troponin I Threonine 144. Role in Myofilament Length–Dependent Activation

Myofilament length–dependent activation is the main cellular mechanism responsible for the Frank–Starling law of the heart. All striated muscle display length-dependent activation properties, but it is most pronounced in cardiac muscle and least in slow skeletal muscle. Cardiac muscle expressing slow skeletal troponin (ssTn)I instead of cardiac troponin (cTn)I displays reduced myofilament length–dependent activation. The inhibitory region of troponin (Tn)I differs by a single residue, proline at position 112 in ssTnI versus threonine at position 144 in cTnI. Here we tested whether this substitution was important for myofilament length–dependent activation; using recombinant techniques, we prepared wild-type cTnI, ssTnI, and 2 mutants: cTnIThr>Pro and ssTnIPro>Thr. Purified proteins were complexed with recombinant cardiac TnT/TnC and exchanged into skinned rat cardiac trabeculae. Force–Ca2+ relationships were determined to derive myofilament Ca2+ sensitivity (EC50) at 2 sarcomere lengths: 2.0 and 2.2 &mgr;m (n=7). Myofilament length-dependent activation was indexed as &Dgr;EC50, the difference in EC50 between sarcomere lengths of 2.0 and 2.2 &mgr;m. Incorporation of ssTnI compared with cTnI into the cardiac sarcomere reduced &Dgr;EC50 from 1.26±0.30 to 0.19±0.04 &mgr;mol/L. A similar reduction also could be observed when Tn contained cTnIThr>Pro (&Dgr;EC50=0.24±0.04 &mgr;mol/L), whereas the presence of ssTnIPro>Thr increased &Dgr;EC50 to 0.94±0.12 &mgr;mol/L. These results suggest that Thr144 in cardiac TnI modulates cardiac myofilament length–dependent activation.

Increased atrial arrhythmia susceptibility induced by intense endurance exercise in mice requires TNFα

Atrial fibrillation (AF) is the most common supraventricular arrhythmia that, for unknown reasons, is linked to intense endurance exercise. Our studies reveal that 6 weeks of swimming or treadmill exercise improves heart pump function and reduces heart-rates. Exercise also increases vulnerability to AF in association with inflammation, fibrosis, increased vagal tone, slowed conduction velocity, prolonged cardiomyocyte action potentials and RyR2 phosphorylation (CamKII-dependent S2814) in the atria, without corresponding alterations in the ventricles. Microarray results suggest the involvement of the inflammatory cytokine, TNFα, in exercised-induced atrial remodelling. Accordingly, exercise induces TNFα-dependent activation of both NFκB and p38MAPK, while TNFα inhibition (with etanercept), TNFα gene ablation, or p38 inhibition, prevents atrial structural remodelling and AF vulnerability in response to exercise, without affecting the beneficial physiological changes. Our results identify TNFα as a key factor in the pathology of intense exercise-induced AF.

Myosin head orientation: a structural determinant for the Frank-Starling relationship

The cellular mechanism underlying the Frank-Starling law of the heart is myofilament length-dependent activation. The mechanism(s) whereby sarcomeres detect changes in length and translate this into increased sensitivity to activating calcium has been elusive. Small-angle X-ray diffraction studies have revealed that the intact myofilament lattice undergoes numerous structural changes upon an increase in sarcomere length (SL): lattice spacing and the I(1,1)/I(1,0) intensity ratio decreases, whereas the M3 meridional reflection intensity (I(M3)) increases, concomitant with increases in diastolic and systolic force. Using a short (∼10 ms) X-ray exposure just before electrical stimulation, we were able to obtain detailed structural information regarding the effects of external osmotic compression (with mannitol) and obtain SL on thin intact electrically stimulated isolated rat right ventricular trabeculae. We show that over the same incremental increases in SL, the relative changes in systolic force track more closely to the relative changes in myosin head orientation (as reported by I(M3)) than to the relative changes in lattice spacing. We conclude that myosin head orientation before activation determines myocardial sarcomere activation levels and that this may be the dominant mechanism for length-dependent activation.

Thick-Filament Strain and Interfilament Spacing in Passive Muscle: Effect of Titin-Based Passive Tension

We studied the effect of titin-based passive tension on sarcomere structure by simultaneously measuring passive tension and low-angle x-ray diffraction patterns on passive fiber bundles from rabbit skinned psoas muscle. We used a stretch-hold-release protocol with measurement of x-ray diffraction patterns at various passive tension levels during the hold phase before and after passive stress relaxation. Measurements were performed in relaxing solution without and with dextran T-500 to compress the lattice toward physiological levels. The myofilament lattice spacing was measured in the A-band (d(1,0)) and Z-disk (d(Z)) regions of the sarcomere. The axial spacing of the thick-filament backbone was determined from the sixth myosin meridional reflection (M6) and the equilibrium positions of myosin heads from the fourth myosin layer line peak position and the I(1,1)/I(1,0) intensity ratio. Total passive tension was measured during the x-ray experiments, and a differential extraction technique was used to determine the relations between collagen- and titin-based passive tension and sarcomere length. Within the employed range of sarcomere lengths (∼2.2-3.4 μm), titin accounted for >80% of passive tension. X-ray results indicate that titin compresses both the A-band and Z-disk lattice spacing with viscoelastic behavior when fibers are swollen after skinning, and elastic behavior when the lattice is reduced with dextran. Titin also increases the axial thick-filament spacing, M6, in an elastic manner in both the presence and absence of dextran. No changes were detected in either I(1,1)/I(1,0) or the position of peaks on the fourth myosin layer line during passive stress relaxation. Passive tension and M6 measurements were converted to thick-filament compliance, yielding a value of ∼85 m/N, which is several-fold larger than the thick-filament compliance determined by others during the tetanic tension plateau of activated intact muscle. This difference can be explained by the fact that thick filaments are more compliant at low tension (passive muscle) than at high tension (tetanic tension). The implications of our findings are discussed.

Titin strain contributes to the Frank-Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

Significance The Frank–Starling law of the heart represents a fundamental regulatory mechanism whereby cardiac pump performance is directly modulated by the extent of diastolic ventricular filling on a beat-to-beat basis. It is now well established that sarcomere length (SL)-induced changes in cardiac contractile protein responsiveness to activating calcium ions play a major role in this phenomenon. However, the molecular mechanisms that underlie this SL-sensing property are not known. Here, we show by small-angle X-ray diffraction and fluorescent probe techniques that the giant protein titin likely transmits the length signal to induce structural alterations in both thin- and thick-filament contractile proteins. These findings provide insights into the molecular basis of the Frank–Starling regulatory mechanism. The Frank–Starling mechanism of the heart is due, in part, to modulation of myofilament Ca2+ sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank–Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.

Nuclear Factor κB Downregulates the Transient Outward Potassium Current Ito,f Through Control of KChIP2 Expression

Rationale: The fast transient outward K+ current (Ito,f) plays a critical role in early repolarization of the heart. Ito,f is consistently downregulated in cardiac disease. Despite its importance, the regulation of Ito,f in disease remains poorly understood. Objective: Because the transcription factor nuclear factor (NF)-&kgr;B is activated in cardiac hypertrophy and disease, we studied the role of NF-&kgr;B in mediating Ito,f reductions induced by hypertrophy. Methods and Results: Culturing neonatal rat ventricular myocytes in the presence of phenylephrine (PE) plus propranolol (Pro), to selectively activate &agr;1-adrenergic receptors, caused reductions in Ito,f, as well as KChIP2 and Kv4.3 expression, while increasing Kv4.2 expression. Inhibition of NF-&kgr;B, via overexpression of a phosphorylation-deficient mutant of I&kgr;B&agr; (I&kgr;B&agr;SA) prevented PE/Pro-induced reductions in Ito,f and KChIP2 mRNA, without affecting Kv4.2 or Kv4.3 expression, suggesting NF-&kgr;B mediates the Ito,f reductions by repressing KChIP2. Indeed, overexpression of the NF-&kgr;B activator I&kgr;B kinase-&bgr; also decreased KChIP2 expression and Ito,f (despite increasing Kv4.2), whereas I&kgr;B&agr;SA overexpression elevated KChIP2 and decreased Kv4.2 levels. In addition, the classic NF-&kgr;B activator tumor necrosis factor &agr; also induced NF-&kgr;B–dependent reductions of KChIP2 and Ito,f. Finally, inhibition of calcineurin did not prevent PE/Pro-induced reductions in KChIP2. Conclusions: NF-&kgr;B regulates KChIP2 and Kv4.2 expression. The reductions in Ito,f observed following &agr;-adrenergic receptor stimulation or tumor necrosis factor &agr; application require NF-&kgr;B–dependent decreases in KChIP2 expression.