2891
J Physiol 588.15 (2010) pp 2891–2903
Length dependence of force generation exhibit similarities between rat cardiac myocytes and skeletal muscle fibres Laurin M. Hanft and Kerry S. McDonald Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Columbia, MO 65212, USA
According to the Frank–Starling relationship, increased ventricular volume increases cardiac output, which helps match cardiac output to peripheral circulatory demand. The cellular basis for this relationship is in large part the myofilament length–tension relationship. Length–tension relationships in maximally calcium activated preparations are relatively shallow and similar between cardiac myocytes and skeletal muscle fibres. During twitch activations length–tension relationships become steeper in both cardiac and skeletal muscle; however, it remains unclear whether length dependence of tension differs between striated muscle cell types during submaximal activations. The purpose of this study was to compare sarcomere length–tension relationships and the sarcomere length dependence of force development between rat skinned left ventricular cardiac myocytes and fast-twitch and slow-twitch skeletal muscle fibres. Muscle cell preparations were calcium activated to yield ∼50% maximal force, after which isometric force and rate constants (k tr ) of force development were measured over a range of sarcomere lengths. Myofilament length–tension relationships were considerably steeper in fast-twitch fibres compared to slow-twitch fibres. Interestingly, cardiac myocyte preparations exhibited two populations of length–tension relationships, one steeper than fast-twitch fibres and the other similar to slow-twitch fibres. Moreover, myocytes with shallow length–tension relationships were converted to steeper length–tension relationships by protein kinase A (PKA)-induced myofilament phosphorylation. Sarcomere length–k tr relationships were distinct between all three cell types and exhibited patterns markedly different from Ca2+ activation-dependent k tr relationships. Overall, these findings indicate cardiac myocytes exhibit varied length–tension relationships and sarcomere length appears a dominant modulator of force development rates. Importantly, cardiac myocyte length–tension relationships appear able to switch between slow-twitch-like and fast-twitch-like by PKA-mediated myofibrillar phosphorylation, which implicates a novel means for controlling Frank–Starling relationships. (Received 24 March 2010; accepted after revision 4 June 2010; first published online 7 June 2010) Corresponding author K. S. McDonald: Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO 65212, USA. Email:
[email protected]
Introduction The heart must have considerable functional plasticity to adjust its output to meet circulatory demand. One way the heart achieves such a large functional capacity is the exquisite sensitivity of ventricular contractility to filling volume. Greater ventricular filling yields increased ventricular stroke output (Lakatta, 1992), providing the heart with an intrinsic mechanism to match cardiac output to circulatory demand on a beat-to-beat basis. This relationship was first described in the early 20th century and is known as the Frank–Starling relationship. The Frank–Starling relationship is modulated by a number of C 2010 The Authors. Journal compilation C 2010 The Physiological Society
factors including autonomic innervation, humoral factors, and exercise; additionally, from a clinical standpoint, the Frank–Starling relationship becomes considerably depressed (in some cases nearly 10-fold) in late stage cardiac failure (Ross & Braunwald, 1964; Holubarsch et al. 1996). The cellular basis for the Frank–Starling relationship is thought to be related, in large part, to the myofilament length–tension relationship. The length–tension relationship is relatively shallow and is similar between cardiac myocytes and skeletal muscle fibres during maximal Ca2+ activations (Gordon et al. 1966; Fabiato & Fabiato, 1975; Moss, 1979). During twitch activations, DOI: 10.1113/jphysiol.2010.190504
2892
L. M. Hanft and K. S. McDonald
which are most analogous to a heartbeat (i.e. thin filaments are not maximally activated), length–tension relationships become considerably steeper in both cardiac and skeletal muscle (Close, 1972; Kentish et al. 1986); however, there has yet to be quantitative comparison of length–tension relationships between cardiac myocytes, fast-twitch skeletal muscle fibres, and slow-twitch skeletal muscle fibres during submaximal Ca2+ activations. The purpose of this study was to compare myofilament length–tension relationships between cardiac myocytes, fast-twitch skeletal muscle fibres, and slow-twitch skeletal muscle fibres during submaximal Ca2+ activations. The experiments were performed on permeabilized single cell preparations to minimize confounding mechanical influences associated with extracellular viscoelastic elements and to provide a direct comparison of myofibrillar length–tension relationships among striated muscle cell types.
J Physiol 588.15
pellet was washed twice with cold relaxing solution, and the skinned cells were then resuspended in 10–20 ml of relaxing solution and kept on ice during the day of the experiment. Skeletal muscle fibre preparation
Skeletal muscle fibres also were obtained from Sprague–Dawley rats anaesthetized by inhalation of isoflurane (20% (v/v) in olive oil) (McDonald, 2000). Fast-twitch and slow-twitch skeletal muscle fibres were obtained from the psoas and soleus muscles, respectively. The muscles were isolated, placed in relaxing solution at 4◦ C and bundles of ∼50 fibres were separated, tied to capillary tubes and stored in relaxing solution containing 50% (v/v) glycerol for up to 4 weeks. Single fibres for mechanical measurements were dissected by gently pulling them from the end of the bundle.
Methods
Experimental apparatus
Experimental animals
The experimental apparatus for physiological measurements of myocyte preparations and skeletal muscle fibres was similar to one previously described in detail (Moss, 1979) and modified for cardiac myocyte preparations (McDonald et al. 1998). Myocyte/fibre preparations were attached between a force transducer and torque motor by placing the ends of the myocyte preparation into stainless steel troughs (25 gauge). The ends of the myocyte/fibre preparations were secured by overlaying a 0.5 mm length of 3-0 monofilament nylon suture (Ethicon, Inc.) onto each end of the myocyte, and then tying the suture into the troughs with two loops of 10-0 monofilament (Ethicon, Inc). The attachment procedure was performed under a stereomicroscope (∼100× magnification) using finely shaped forceps. Prior to mechanical measurements the experimental apparatus was mounted on the stage of an inverted microscope (model IX-70, Olympus Instrument Co., Japan), which was placed upon a pneumatic vibration isolation table having a cut-off frequency of ∼1 Hz. Mechanical measurements were performed using a capacitance-gauge transducer (Model 403, sensitivity of 20 mV mg−1 (plus a 10× amplifier for cardiac myocytes) and resonant frequency of 600 Hz; Aurora Scientific, Inc., Aurora, ON, Canada). Length changes were introduced using a DC torque motor (model 308, Aurora Scientific, Inc.) driven by voltage commands from a personal computer via a 12-bit D/A converter (AT-MIO-16E-1, National Instruments Corp., Austin, TX, USA). Force and length signals were digitized at 1 kHz and stored on a personal computer using LabView for Windows (National Instruments Corp.). Sarcomere length was monitored simultaneously with force and length measurements using
Male Sprague–Dawley rats (6 weeks old) were obtained from Harlan (Madison, WI, USA), housed in groups of two, and provided with access to food and water ad libitum. A group of animals was provided with propranolol (1 mg ml−1 )-treated water for 3–5 days. Propranolol is a β-adrenergic antagonist and was administered to minimize baseline PKA-induced phosphorylation of myofilament proteins and to increase the probability of obtaining a myocyte preparation that exhibited a shallow sarcomere length–tension relationship. All procedures involving animal use were performed according to protocols that were reviewed and approved by the Animal Care and Use Committee of the University of Missouri, and the experiments comply with the policies of The Journal of Physiology as set out by Drummond (2009).
Cardiac myocyte preparation
Myocytes were obtained by mechanical disruption of rat hearts (n = 25) (McDonald, 2000). Rats were placed in a small air-tight chamber and anaesthetized by inhalation of isoflurane (20% (v/v) in olive oil), and their hearts were quickly removed and placed in ice-cold relaxing solution. The atria and right ventricles were removed and left ventricles were cut into 2–3 mm pieces and further disrupted for 5–10 s using a Waring blender. The resulting suspension of cells and cell fragments were centrifuged for 105 s at 165 g. The myocytes were subsequently skinned by suspending the pellet for 4 min in 0.3% ultrapure Triton X-100 (Pierce Biotechnology, Inc., Rockford, IL, USA) in relaxing solution (composition given below). The
C 2010 The Authors. Journal compilation C 2010 The Physiological Society
J Physiol 588.15
Cardiac myocyte length–tension relationships
IonOptix SarcLen system (IonOptix, Milton, MA, USA), which used a fast Fourier transform algorithm of the video image of the myocyte. Microscopy was done using a 40× objective (Olympus UWD 40) and a 2.5× intermediate lens. Solutions
Compositions of relaxing and activating solutions used in mechanical measurements were as follows: 7 mM EGTA, 1 mM free Mg2+ , 20 mM imidazole, 4 mM MgATP, 14.5 mM creatine phosphate, pH 7.0, various Ca2+ concentrations between 10−9 M (relaxing solution) and 10−4.5 M (maximal Ca2+ activating solution), and sufficient KCl to adjust ionic strength to 180 mM. The final concentrations of each metal, ligand and metal–ligand complex at 13◦ C were determined with the computer program of Fabiato (1988). Preceding each Ca2+ activation, myocyte preparations were immersed for 30 s in a solution of reduced Ca2+ -EGTA buffering capacity, which was identical to normal relaxing solution except that EGTA was reduced to 0.5 mM. This protocol resulted in more rapid development of steady state force during subsequent activation and helped preserve the striation pattern during activation. Relaxing solution in which the ventricles were mechanically disrupted and myocytes and skeletal muscle fibres were resuspended contained 2 mM EGTA, 5 mM MgCl2 , 4 mM ATP, 10 mM imidazole, and 100 mM KCl at pH 7.0 with the addition of a protease inhibitor cocktail (Set I, Calbiochem, San Diego, CA, USA). Sarcomere length–tension measurements
All mechanical measurements on cardiac myocytes and skeletal muscle fibres were performed at 13 ± 1◦ C. For mechanical measurements on myocytes, a preparation was chosen from a cell suspension based on two morphological criteria: (i) the myocyte preparation had to be at least 100 μm in length when floating free, which allowed enough size to pick up with forceps and enough length to secure in the troughs by placing suture over ∼10–20 μm of each of its ends, and (ii) the preparation needed to be rod-shaped with limited or no branching. Following attachment, the relaxed preparation was adjusted to a sarcomere length of ∼2.35 μm and then the preparation was maximally Ca2+ activated in pCa 4.5 solution. For sarcomere length–tension measurements the cell preparation was transferred to a pCa solution that yielded ∼50% maximal (i.e. pCa 4.5 or P4.5 ) force and then isometric force was measured over a range of sarcomere lengths monitored by the IonOptix SarcLen system (Fig. 1). Isometric force and sarcomere length were measured simultaneously. Sarcomere length was adjusted between ∼2.35 μm and sometimes down to ∼1.4 μm by C 2010 The Authors. Journal compilation C 2010 The Physiological Society
2893
manual manipulation of the length micrometer while the preparation was Ca2+ activated. Striation spacing below ∼1.6 μm (the length of the thick filament) was readily discernable during submaximal Ca2+ activations and has been reported previously for rabbit skinned skeletal muscle fibres (Allen & Moss, 1987), rat cardiac trabeculae (Kentish & Stienen, 1994), and rat cardiac myocytes (Fabiato & Fabiato, 1975; Bardswell & Kentish, 2003). After each sarcomere length change, ∼10–15 s were allowed for development of steady-state force. Force at each sarcomere length was obtained via a slack-restretch manoeuvre (see below for description). For analysis, force at each sarcomere length was normalized to the force obtained at sarcomere length ∼2.35 μm (during the sub-maximal Ca2+ activation). Since force during submaximal Ca2+ activations invariably rose slightly during the sustained activation, normalized forces were calculated by interpolating force measurements at sarcomere length 2.35 μm, which were performed at the beginning and end of the series of force measurements. At the end of each experiment, preparations were activated a second time in pCa 4.5 solutions and only experiments in which maximal tension remained >90% of initial were used for analysis. To assess the effects of PKA, length–tension relationships were performed before and after 45 min incubation with PKA (Sigma, 0.125 U μl−1 ). The pCa solution for length–tension curves was adjusted from 5.80 ± 0.09 to 5.68 ± 0.04 to yield the same forces before and after PKA due to decreased Ca2+ sensitivity of force following PKA. Control experiments included sarcomere length–tension measurements before and after incubation with PKA in the presence of protein kinase inhibitor (PKI) (300 μg ml−1 ). Since PKI inhibits PKA activity, these control experiments tested the specific effects of PKA-induced myofilament phosphorylation. The range of pCa solutions used to obtain ∼50% of P 4.5 was pCa 6.0 for fast-twitch fibres, pCa 6.1–6.0 for slow-twitch fibres, and pCa 6.0–5.7 for cardiac myocyte preparations. For PKA experiments in cardiac myocyte preparations, pCa 5.9–5.7 was used pre-PKA and pCa 5.7–5.6 post-PKA. Measurement of the rate of force redevelopment
The kinetics of force redevelopment were obtained using a procedure previously described for skinned cardiac myocyte preparations (Korte et al. 2003; Hinken & McDonald, 2004; Hanft & McDonald, 2009). While in Ca2+ activating solution, the myocyte preparation was rapidly shortened by 15–20% of initial length (Lo ) to yield zero force. The myocyte preparation was then allowed to shorten for ∼20 ms; after 20 ms the preparation was rapidly re-stretched to ∼105% of its initial length (Lo ) for 2 ms and then returned to Lo . The slack–restretch manoeuvre is thought to dissociate
2894
L. M. Hanft and K. S. McDonald
cross-bridges, so that subsequent force redevelopment arises from cross-bridge reattachment and transition to force-generating states. The slack-restretch manoeuvre was performed to measure Ca2+ activated force at each sarcomere length and thus allowed a thorough characterization of the sarcomere length dependence of the rate of force development. Tension redevelopment following a slack–restretch manoeuvre was fitted by a single exponential equation: F = F max (1 − e−ktr t ) + F res
(1)
where F is tension at time t, F max is maximal tension, k tr is the rate constant of force development, and F res represents any residual tension immediately after the slack–restretch manoeuvre.
J Physiol 588.15
SDS-PAGE and autoradiography
To examine myofibrillar substrates of PKA, myofibrillar samples from control and propranolol treated rats were incubated with the catalytic subunit of PKA in the presence of radiolabelled ATP, separated by SDS-PAGE, and visualized by autoradiography. Briefly, 10 μg of skinned cardiac myocytes were incubated with the catalytic subunit of PKA (2.5 U μl−1 ) and 50 μCi [γ-32 P]ATP for 30 min. As a control experiment, cardiac myocytes also were incubated with PKA in the presence of PKI (4.4 U μl−1 ). The reaction was stopped by the addition of electrophoresis sample buffer and heating at 95◦ C for 3 min. The samples were then separated by SDS-PAGE (12% acrylamide), silver stained, dried and subsequently exposed to X-ray film for ∼24 h at −70◦ C.
Figure 1. Simultaneous measurement of force at three different sarcomere lengths in a cardiac myocyte preparation Top right, a photomicrograph of a cardiac myocyte at sarcomere length 2.30 μm and an illustration of the permeabilized muscle cell attachment apparatus. The rest of the figure shows a slow-time base sarcomere length recording using an IonOptix SarcLen system (IonOptix, Milton, MA, USA) and the corresponding (fast-time base) force traces at sarcomere lengths 2.30 μm, 2.15 μm and 1.95 μm during submaximal Ca2+ activation (pCa 5.7) of a rat cardiac myocyte preparation. Upper left force trace is during maximal calcium activation (pCa 4.5) at sarcomere length 2.30 μm. C 2010 The Authors. Journal compilation C 2010 The Physiological Society
Cardiac myocyte length–tension relationships
J Physiol 588.15
2895
Table 1. Striated muscle cell preparation characteristics
Fast-twitch fibres Slow-twitch fibres Cardiac myocytes
n
Length (μm)
Width (μm)
Maximum force (P 4.5 ) (kN m−2 )
pCa for P/P 4.5
P/P 4.5
10 8 19
1040 ± 238 1034 ± 154 115 ± 30
124 ± 18 91 ± 11 20 ± 5
119 ± 32 133 ± 30 71 ± 24
6.0 6.05 ± 0.05 5.86 ± 0.08
0.52 ± 0.07 0.47 ± 0.11 0.53 ± 0.11
Values are means ± S.D. P/P 4.5 , fraction of maximum isometric force (P 4.5 ). pCa for P/P 4.5 , pCa solution used for sarcomere length–tension relationships.
Results
The characteristics of the fast-twitch skeletal muscle fibres, slow-twitch skeletal muscle fibres, and cardiac myocyte preparations are shown in Table 1. Slopes of length–tension relationships were determined by linear regression and compared between muscle cell types by one-way ANOVA. A Student–Neuman–Keuls post hoc test assessed differences between means. Slopes before and after PKA treatment were compared by Student’s t test for paired data. All values are means ± S.D. unless otherwise indicated. P < 0.05 was chosen to indicate statistical significance.
Sarcomere length–tension relationships in striated muscle cell types
Passive Tension (kN/m2)
Statistical analysis
1.2
Fast-Twitch Fibers Slow-Twitch Fibers Cardiac Myocytes
2.0
1.0
Relative Force
9 8 7 6 5 4 3 2 1 0
Sarcomere length–tension relationships were first compared between fast-twitch and slow-twitch skeletal muscle fibres during submaximal Ca2+ activations. Fast-twitch skeletal muscle fibres exhibited a relatively steep length–tension relationship while slow-twitch skeletal muscle fibres exhibited a relatively shallow sarcomere length–tension relationship (Fig. 2), which was expected based upon previous measures of length–tension relationships at submaximal Ca2+ activations (Allen &
2.2 2.4 2.6 Sarcomere Length (μm)
2.8
0.8 0.6 Fast-Twitch Fibers Slow-Twitch Fibers Cardiac Myocytes - Shallow Cardiac Myocytes - Steep
0.4 0.2 0.0 1.2
1.4
1.6
1.8
2.0
2.2
2.4
Sarcomere Length (μm) Figure 2. Sarcomere length–tension relationships for rat fast-twitch skeletal muscle fibres, slow-twitch skeletal muscle fibres and cardiac myocytes Cardiac myocyte preparations exhibited two populations of length–tension relationships, one steeper even than fast-twitch fibres and the other similar to slow-twitch fibres. Inset shows passive length–tension relationships of rat skinned cardiac myocytes, fast-twitch skeletal muscle fibres, and slow-twitch skeletal muscle fibres. The steepness of active length–tension relationships was independent of passive tension. Slopes of length–tension relationships were: fast-twitch fibres: 1.62 ± 0.17∗ # (n = 10); slow-twitch fibres: 1.02 ± 0.12§ (n = 8); cardiac myocytes (steep population): 1.85 ± 0.16∗ #§ (n = 6); cardiac myocytes (shallow population): 1.09 ± 0.15§ (n = 13); ∗ P < 0.05 vs. slow-twitch fibres, #P < 0.05 vs. cardiac myocyte shallow population, §P < 0.05 vs. fast-twitch fibres. C 2010 The Authors. Journal compilation C 2010 The Physiological Society
2896
L. M. Hanft and K. S. McDonald
Moss, 1987) and length dependence of calcium sensitivity of force (Allen & Moss, 1987; McDonald et al. 1997; Konhilas et al. 2002). Cardiac myocyte preparations, on the other hand, exhibited two populations of length–tension relationships, one steeper even than fast-twitch fibres and the other similar to slow-twitch fibres (Fig. 2). The active sarcomere length–tension relationships were unrelated to passive force levels as cardiac myocytes exhibited just one population of sarcomere length–passive tension relations, which was considerably steeper than both fast-twitch and slow-twitch skeletal muscle fibres (Fig. 2 inset). Specifically, passive tension was 3.5 ± 2.5 kN m−2 at 2.30 ± 0.04 μm in cardiac myocyte preparations, which was much greater than fast-twitch (1.1 ± 0.8 kN m−2 at 2.70 ± 0.07 μm) and slow-twitch (1.4 ± 0.5 kN m−2 at 2.70 ± 0.07 μm) skeletal muscle fibres.
J Physiol 588.15
Modulation of cardiac myocyte length–tension relationships by PKA-induced myofilament phosphorylation
Since β-adrenergic stimulation is known to shift the left ventricular Frank–Starling relationship upward (i.e. steeper) we next addressed whether phosphorylation of myofibrillar proteins by PKA (a downstream mediator of β-adrenergic signalling) would regulate sarcomere length–tension relationships. Treatment of cardiac myocytes with PKA caused the phosphorylation of two myofibrillar proteins, myosin binding protein-C (MyBP-C) and cardiac troponin I (cTnI) (Fig. 3A). PKA treatment of cardiac myocytes (with shallow, slow-twitch-like relationships) steepened length–tension relationships to yield relationships more
Figure 3. Effects of PKA on cardiac myocyte length–tension relationships A, an autoradiogram showing radiolabelled phosphate incorporation into rat cardiac myofibrillar proteins (MyBP-C and cTnI) upon PKA treatment. Lanes 1 and 2 contain permeabilized cardiac myocytes in the presence of [32 P]ATP either without (lane 1) or with PKA (lane 2). B, cardiac myocyte force traces during submaximal Ca2+ activations at four different sarcomere lengths before and after PKA treatment. C, normalized cardiac myocyte sarcomere length–tension relationships before and after PKA treatment (n = 6). PKA-induced phosphorylation markedly steepened the length–tension relationship in these preparations. Left inset shows a representative experiment whereby PKA treatment did not alter the length–tension curve of a cardiac myocyte preparation that had a steep length–tension relationship. Right inset shows a control experiment whereby the addition of PKA in the presence of protein kinase inhibitor (PKI) did not alter the length–tension relationship. The bottom inset autoradiogram shows enhanced PKA-induced phosphate incorporation into cardiac myofilament proteins from rats treated with propranolol (lane 2) versus control rats (lane 1). PKI markedly inhibited the PKA-induced phosphate incorporation into cardiac myofibrillar proteins (lane 3). C 2010 The Authors. Journal compilation C 2010 The Physiological Society
Cardiac myocyte length–tension relationships
J Physiol 588.15
similar to fast-twitch skeletal muscle fibres (Fig. 3C). (Length–tension slope constants were: cardiac myocytes before PKA: 1.02 ± 0.08; cardiac myocytes after PKA: 1.75 ± 0.43∗ (n = 6); ∗ P < 0.05 vs. before PKA.) In contrast, PKA did not alter length–tension relationships in myocyte preparations that already exhibited steep relationships prior to PKA treatment (Fig. 3C, left inset). When PKA was added to cardiac myocytes in the presence of protein kinase inhibitor (PKI) (Fig. 3C right inset) there was a marked reduction in PKA-induced phosphorylation of MyBP-C and cTnI (Fig. 3C autoradiogram) and the length–tension relationship was unaltered, implicating PKA-mediated specific phosphorylation(s) as the mechanism underlying the steepening of length–tension relationships. Histogram of slopes indicates bimodal variation in striated muscle cell length–tension relationships
Figure 4 shows a histogram of the slopes of length–tension relationships for all the muscle cell preparations. The histogram indicates that there are two populations of length–tension relationships in striated muscle. One population has shallow slopes that ranged from 0.80 to 1.30 and consists of slow-twitch skeletal muscle fibres and a sub-group of cardiac myocytes. The other population consisted of fast-twitch fibres and a different sub-group of cardiac myocytes exhibiting steeper slopes that range from 1.40 to 2.40. Regarding cardiac myocytes, our initial sampling of left ventricular myocytes yielded an approximate 50:50 distribution of steep and shallow
length–tension relationships. Specifically, of the 13 cardiac myocyte preparations chosen from control rat hearts six exhibited a steep sarcomere length–tension relationship (slope ≥1.67) and seven had a shallow length–tension relationship (slope ≤1.34). Next, since we hypothesized that endogenous levels of PKA-mediated protein phosphorylation are responsible for determining the slope of the length–tension relationship, we treated six rats with the β-adrenergic blocker propranolol to reduce basal levels of PKA-induced phosphorylation of MyBP-C and cTnI (Fig. 3C autoradiogram). From these myocyte suspensions, 6 of 6 preparations exhibited shallow slopes (≤1.17). All six of these preparations exhibited steeper slopes following PKA treatment. The reason for this distribution pattern is unclear but may arise from regional differences across the ventricle (e.g. epicardial versus endocardial; Ait mou et al. 2008). We tested this idea by performing PKA-induced back-phosphorylation assays on samples from epicardial, mid-wall and endocardial regions of the left ventricle. We did not observe any significant differences in PKA-induced back-phosphorylation signals between regions implicating similar levels of basal PKA phosphorylation levels (Fig. 5). Given these results and since we observed a near 50:50 distribution in control ventricles, these results implicate near-neighbour cellular differences, perhaps due to variable sympathetic innervation or sympathetic responsiveness.
Fast-Twitch Fibers Slow-Twitch Fibers Cardiac Myocytes - Shallow Cardiac Myocytes - Steep Cardiac Myocytes - After PKA
9
Number of observations
2897
8 7 6 5 4 3 2 1 0
0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4
SL-Tension Slopes Figure 4. Histogram of slopes of sarcomere length–tension relationships The histogram indicates two populations of sarcomere length–tension relationships in striated muscle. One population had shallow slopes (ranging from 0.80 to 1.30) and consisted of slow-twitch skeletal muscle fibres and a subgroup of cardiac myocytes. The other population consisted of fast-twitch fibres and a different subgroup of cardiac myocytes that had steeper slopes that ranged from 1.40 to 2.40. C 2010 The Authors. Journal compilation C 2010 The Physiological Society
Figure 5. Autoradiogram showing PKA-mediated phosphorylation of MyBP-C and cTnI of myofibrils from different regions of rat ventricular free wall PKA-induced back-phosphorylation of myofibrils did not show any differences from myocytes isolated from the epicardium, mid-wall, or endocardial regions of the left ventricle.
2898
L. M. Hanft and K. S. McDonald
was examined in cardiac myocyte preparations (Fig. B). PKA treatment slowed force redevelopment rates at all sarcomere lengths during the submaximal Ca2+ activation (consistent with our previous report; Hanft & McDonald, 2009) and also eliminated the noticeable increase in k tr as sarcomere length was shortened to less than ∼1.85 μm that was observed in all the other preparations.
Sarcomere length dependence of the rate of force development
Rates of force development were examined over a large range of sarcomere lengths during submaximal Ca2+ activations of fast-twitch fibres, slow-twitch fibres and cardiac myocyte preparations (Fig. 6A). For fast-twitch fibres the rate constant of force development (k tr ) fell as sarcomere length decreased from ∼2.35 μm to ∼1.90 μm. However, as sarcomere length was further reduced (from ∼1.90 μm to ∼1.60 μm) there was a large progressive increase in k tr values. Slow-twitch skeletal muscle fibres exhibited a slight progressive increase in k tr as sarcomere length was reduced from ∼2.35 μm to ∼1.90 μm; this was followed by a somewhat larger progressive increase in k tr as sarcomere length further shortened towards ∼1.50 μm. Cardiac myocyte preparations that exhibited a shallow length–tension relationship displayed a subtle U-shaped pattern in the variation in k tr as a function of sarcomere length in the range ∼2.30 to ∼1.50 μm. Cardiac myocytes with steep length–tension relationships displayed a nearly flat sarcomere length dependence of k tr from ∼2.30 μm to ∼1.85 μm followed by an increase in k tr at sarcomere lengths less than ∼1.85 μm. Interestingly, this rise in k tr at short sarcomere was somewhat similar to that observed in fast-twitch skeletal muscle fibres. The effects of PKA treatment on the sarcomere length dependence of k tr
A 7
Fast-Twitch Fibers Slow-Twitch Fibers Cardiac Myocytes - Shallow Cardiac Myocytes - Steep
Discussion Our results indicate that there are two populations of cardiac myocytes with regards to length–tension relationships, one steep like fast-twitch skeletal muscle fibres and the other shallow like slow-twitch fibres. In addition, cardiac myocyte preparations with shallow length–tension relationships shifted to steeper (fast-twitch-like) length–tension relationships following PKA-induced phosphorylation of MyBP-C and cTnI, which implicates the β-adrenergic system as an important control mechanism for myocyte length–tension relationships and, thus, the Frank–Starling mechanism. The key determinant of the Frank–Starling relationship in cardiac muscle is the sarcomere length–tension relationship in striated muscle. The ascending limb of the length–tension relationship is relatively shallow and is similar in cardiac myocytes and skeletal muscle fibres
B
7
5
5 ktr (sec-1)
Cardiac Myocytes – Before PKA Cardiac Myocytes - After PKA
6
6
4
4
3
3
2
2
1
1
0 1.4
J Physiol 588.15
1.5
1.6
1.7
1.8
1.9
2.0
2.1
Sarcomere Length (μm)
2.2
2.3
2.4
0 1.4
1.5
1.6
1.7
1.8
1.9
2.0
2.1
2.2
2.3
2.4
Sarcomere Length (μm)
Figure 6. Sarcomere length dependence of rate constant of force redevelopment (k tr ) A, sarcomere length dependence of ktr for fast-twitch skeletal muscle fibres, slow-twitch fibres and the two populations of cardiac myocyte preparations, one that displayed steep and the other that displayed shallow length–tension relationships. Sarcomere length dependence of ktr was unique for each of the different muscle cell preparations and ktr seemed to increase at sarcomere lengths below ∼1.85 μm in all the preparations. This occurred despite reductions in force, implicating that sarcomere length overrides the activation dependence of ktr normally observed in striated muscle preparations (Gordon et al. 2000). B, sarcomere length dependence of ktr in cardiac myocyte preparations before and after PKA treatment. PKA decreased ktr at all sarcomere lengths and prevented the up-turn in ktr sarcomere lengths below ∼1.85 μm observed in all other preparations. (Data points are means ± S.E.M.) C 2010 The Authors. Journal compilation C 2010 The Physiological Society
J Physiol 588.15
Cardiac myocyte length–tension relationships
during maximal Ca2+ activations (Gordon et al. 1966; Fabiato & Fabiato, 1975) and has been shown to be due to mechanical and structural factors at the level of the sarcomere as opposed to variations in Ca2+ release (Moss, 1979). During twitch or submaximal Ca2+ activations, length–tension relationships become steeper in both skeletal (Close, 1972) and cardiac muscle (Fabiato & Fabiato, 1975; Kentish et al. 1986). The steeper relationship appears to be due, at least in part, to mechanical/structural factors such as changes in lattice spacing or altered cross-bridge orientation, which tend to reduce the likelihood of cross-bridge binding and thin filament activation at progressively lower sarcomere lengths (for review see Fuchs & Martyn, 2005). The sarcomere length dependence of Ca2+ sensitivity of tension has been compared between muscle types in previous studies. For those experiments, steady state tension–pCa relationships were measured at a long sarcomere length (∼2.3 μm) and at a short sarcomere length (∼2.0 μm); this provides a quantitative measure of the responsiveness of the tension–pCa relationship in a muscle to a significant change in sarcomere length. McDonald et al. (1997) found that sarcomere length dependence of Ca2+ sensitivity of tension was considerably greater in rabbit fast-twitch skeletal muscle fibres (pCa50 = 0.24) compared to rat slow-twitch skeletal muscle fibres (pCa50 = 0.10). Konhilas et al. (2002) compared rat fast-twitch skeletal muscle fibres, rat slow-twitch skeletal muscle fibres, and rat cardiac trabeculae and found sarcomere length dependence of Ca2+ sensitivity of tension to be ordered as cardiac trabeculae > fast-twitch skeletal muscle fibres > slow-twitch skeletal muscle fibres. In the current study, we expanded the measurements to include a full range of sarcomere lengths in the ascending limb and compared rat single fast-twitch and slow-twitch skeletal muscle fibres with rat cardiac myocyte preparations to avoid effects due to extracellular components associated with multicellular preparations, which may generate recoil forces that would tend to accentuate length-dependent activation. Consistent with previous reports, fast-twitch skeletal muscle fibres exhibited greater length dependence of tension than slow-twitch skeletal muscle fibres. In addition, we found a population of cardiac myocytes that exhibited slightly steeper length–tension relationship than even fast-twitch skeletal muscle fibres, which is consistent with the finding of Konhilas et al. (2002). However, a novel finding was a population of cardiac myocytes that had very shallow length–tension relationships, and in fact, the relationships were the same as those in slow-twitch skeletal muscle fibres. Our identification of two populations of cardiac myocyte length–tension relationships suggests a possible mechanism for the recent finding of differences in length dependence of Ca2+ sensitivity of tension in guinea pig myocardium obtained from either the subendocardium or subepicardium C 2010 The Authors. Journal compilation C 2010 The Physiological Society
2899
regions of the left ventricular free wall (Ait mou et al. 2008). A key question that still remains in the field is why there are such discernable differences in length–tension relationships between fast-twitch and slow-twitch skeletal muscle fibres. A definitive answer to this question would necessitate systematic replacement of putative molecules that cause these differences. For example, nearly complete exchange of the skeletal isoform of troponin C (sTnC) with cardiac troponin C (cTnC) did not alter the length dependence of Ca2+ sensitivity of force, implicating that TnC isoforms are neither necessary nor sufficient to explain length–tension differences between fast-twitch and slow-twitch fibres (Moss et al. 1991). Other molecules including TnI, troponin T (TnT), tropomyosin (Tm), MyBP-C, myosin light chains, myosin heavy chains, and titin are all potential targets that could be experimentally tested as important molecules involved in yielding length–tension differences by use of either in vitro replacement strategies or genetic manipulation. The work in this study did not focus on defining the proteins that mediate differences between fast-twitch and slow-twitch fibres, but the comparison of length–tension relationships between cardiac myocytes and the two skeletal muscle fibre types seemed to uncover an important factor that might contribute to length dependent differences. The data suggest that variations in the positive cooperativity in the activation of force development could, at least in part, define length–tension characteristics. Along these lines, PKA-induced phosphorylation of cardiac myofilaments caused a shift in length–tension relationships from slow-twitch-like to fast-twitch-like. PKA-mediated phosphorylation also has been shown to increase the Hill coefficient of tension–pCa relationships in failing human skinned cardiac myocytes (van der Velden et al. 2000), permeabilized rat trabeculae (Konhilas et al. 2003) and rat cardiac myocyte preparations (Hanft & McDonald 2010); Hill coefficients are a supposed indicator of positive cooperativity of force activation. Mechanistically, with greater positive cooperativity of thin filament activation (as seen in fast-twitch fibres and in PKA-treated cardiac myocytes) a small increase in sarcomere length would tend to enhance recruitment of non-cycling cross-bridges into the pool of cycling force generating cross-bridges, leading to a steeper rise in force with a relatively small increase in sarcomere length. This idea is consistent with the much greater length dependence of Ca2+ sensitivity of force observed in highly cooperative fast-twitch fibres compared to less cooperative slow-twitch fibres (McDonald et al. 1997; Konhilas et al. 2002). As mentioned above, the exact molecules involved in manifestation of these differences in cooperative activation of force are unclear but most likely there is an array of molecules working in a highly orchestrated manner and our results showing that PKA-mediated cardiac
2900
L. M. Hanft and K. S. McDonald
myofilament phosphorylation steepens length–tension relationships suggests cTnI and/or MyBP-C are involved in this process. In addition to differences in sarcomere length–tension relationships between striated muscle types, there also were clear differences in sarcomere length dependence of k tr between muscle types (Fig. 6A). Fast-twitch fibres exhibited slower rates of force development with shorter sarcomere lengths but then rates sharply increased at sarcomere lengths below ∼1.90 μm. Interestingly, this pattern was similar to the activation dependence of k tr for fast-twitch fibres in the presence of 6 μM NEM-S1 (Fitzsimons et al. 2001a). This may implicate a similar mechanism limiting the rate of force development, which for the NEM-S1 result was interpreted using a model in which strongly binding cross-bridges recruit non-cycling cross-bridge into the pool of cycling cross-bridges, whereby at low levels of Ca2+ activation NEM-S1 has presumably saturated the recruitment pool thereby speeding force development since recruitment is the rate-limiting step in this model (Campbell, 1997; Fitzsimons et al. 2001a). A similar phenomenon (i.e. fewer cross-bridges available for recruitment) may occur at very short sarcomere lengths due to, perhaps, less than optimal lattice spacing (or cross-bridge orientation) and/or reduced near-neighbour regulatory unit recruitment as a result of reduced strain on titin and/or thin filaments as previously suggested by Korte & McDonald (2007). Slow-twitch fibres exhibited faster rates of force development as sarcomere length decreased, again suggesting a reduction in the cooperative spread of thin activation at short sarcomere lengths. Cardiac myocytes exhibited lesser changes in k tr values with changes in sarcomere length especially from ∼2.30 μm to 1.90 μm, perhaps indicating that near-neighbour cooperative spread of thin activation is less instrumental in regulating force development rates in cardiac muscle (Gillis et al. 2007; Kreutziger et al. 2008), at least in response to this sarcomere length range and with a constant activator [Ca2+ ]. Another interesting aspect of the results is that sarcomere length clearly over-rode the Ca2+ activation dependence of k tr previously reported for slow-twitch (Metzger & Moss, 1990), fast-twitch (Brenner, 1988; Metzger et al. 1989; Metzger & Moss, 1990; Sweeney & Stull, 1990; Swartz & Moss, 1992; Regnier et al. 1999; Fitzsimons et al. 2001a; Gillis et al. 2007), and cardiac muscles (Wolff et al. 1995; Vannier et al. 1996; Fitzsimons et al. 2001b; Adhikari et al. 2004; Gillis et al. 2007). This suggests that sarcomere length per se elicits alterations of thick and thin filament properties in addition to the effects of Ca2+ activation alone. Defining these differences will require additional work but should provide important insights into overall understanding of the factors that regulate striated muscle contraction. As discussed above, length–tension relationships in cardiac muscle become steeper with decreased levels
J Physiol 588.15
of Ca2+ activation (Fabiato & Fabiato, 1975). Interestingly, though, the Frank–Starling relationship becomes quite steep following β-adrenergic stimulation (Guyton et al. 1973); this occurs despite augmented intracellular Ca2+ , which would tend to reduce the steepness of the length–tension relationship. This implies that there is another mechanism, independent of Ca2+ -sensitivity, by which the Frank–Starling mechanism is regulated at the myofilament level. One possibility is phosphorylation of myofilament proteins by the β-adrenergic signalling cascade, which would steepen the length–tension relationship and also could yield the observed heterogeneous length–tension curves among cardiac myocytes at rest given the non-uniform distribution of sympathetic nerve endings in the mammalian heart. Consistent with this idea, we found that PKA-induced phosphorylation of myofibrillar proteins caused a steepening of sarcomere length–tension relationship in cardiac myocytes that previously had shallow length–tension relationships. One caveat in relation to our finding that PKA-mediated phosphorylation increased length dependence of force was that a higher concentration of activator Ca2+ was used after PKA treatment. Greater activator [Ca2+ ] was needed to obtain the same force level as in control myocardium due to the well characterized decrease in Ca2+ sensitivity of force following PKA-induced phosphorylation of myofilament proteins (Solaro, 1986). We normalized force before and after PKA because of the previously reported force dependence of sarcomere length–tension relationships (Fabiato & Fabiato, 1975), i.e. if activator Ca2+ had been kept constant there would have been less force after PKA, which would have biased the measurements toward steeper sarcomere length–tension curves. However, it also is possible that greater activator calcium could augment cooperative activation of force by Ca2+ occupancy of more regulatory units (i.e. 1 troponin, 1 tropomyosin and 7 actins) and, thus, increasing their probability of activation (i.e. increased probability of recruitment of cross-bridges from the non-cycling pool to the cycling, force generating pool) (Regnier et al. 2004; Gillis et al. 2007). As mentioned above, increased cooperativity in thin filament activation may be a mechanism to elicit steeper length–tension curves. The exact contribution of Ca2+ per se compared to Ca2+ -activated level of force on sarcomere length–tension relationships cannot be determined clearly from our studies. However, it is clear that PKA-induced phosphorylation of myofibrillar proteins is able to override the well-established shallowing of length–tension curves as activator [Ca2+ ] is increased (Fabiato & Fabiato, 1975), emphasizing the importance of this post-translational modification in mediating a key cellular mechanism thought to underlie the Frank–Starling relationship. Future experiments will address whether C 2010 The Authors. Journal compilation C 2010 The Physiological Society
J Physiol 588.15
Cardiac myocyte length–tension relationships
post-translational modification (i.e. phosphorylation status) of MyBP-C or cTnI is most important in mediating length–tension relationships in cardiac myocytes. These experiments will involve protein exchange or use of transgenic animals with altered myofibrillar proteins. These approaches are necessary given that use of commercially available cardiac phosphatases yields non-specific removal of phosphate groups from myofibrillar proteins, which precludes meaningful mechanistic interpretation. Interestingly, PKA-induced phosphorylation of cardiac myofilaments also has been reported to yield several other responses that shift cardiac myocytes toward the functional capabilities of fast-twitch skeletal muscle fibres. These include increased cooperativity of myofilament activation (Konhilas et al. 2003), faster cross-bridge cycling and myocyte shortening (Strang et al. 1994; Hanft & McDonald, 2009) and faster relaxation (Kentish et al. 2001; Takimoto et al. 2004). All these functional properties contribute to the more explosive and powerful contractions of fast-twitch muscle. Thus, the shift in cardiac myocytes toward fast-twitch-like properties most likely helps produce greater stroke volume (i.e. increased contractility) following β-adrenergic stimulation. In addition, the steepening of the length–tension relationship with PKA-induced phosphorylation (as reported in this study) is likely to be an important way to facilitate the relaxation of active tension at the shorter sarcomere lengths associated with end systole. This is especially important given that β-adrenergic stimulation is associated with higher [Ca2+ ], which (as mentioned above) would tend to reduce the steepness of the length–tension relationship. Such a myofibrillar mechanism to augment force reduction at short sarcomere lengths could be very important physiologically as a means to accelerate relaxation in the face of shorter systolic and diastolic times associated with higher heart rates. In contrast, shallow cardiac myocyte length–tension relationships (like that of slow-twitch skeletal muscle) may be advantageous as a way to optimize stroke volume over a wide range of preloads to tune cardiac output to peripheral circulatory demands. Cardiac myocytes exhibiting shallow length–tension relationships would prolong the systolic period and delay relaxation due to the maintenance of force generating cross-bridges, even at short sarcomere lengths. Certainly, at lower heart rates associated with rest there is less need for early relaxation and/or to speed relaxation given the longer diastolic time available for ventricular filling. Expression of such a range of length–tension capabilities in cardiac myocytes provides a wide dynamic range in contractility in response to variations in the demands placed upon the heart. This myofibrillar functional range could explain, in large part, the well-defined steepening of the Frank–Starling relationship with sympathetic stimulation (Guyton et al. 1973). Consistent with this idea, the β-adrenergic signalling system is down-regulated in failing C 2010 The Authors. Journal compilation C 2010 The Physiological Society
2901
hearts (Bristow et al. 1982), which exhibit downward and rightward shifts in the Frank–Starling relationship (i.e. shallow Frank–Starling curves) (Braunwald & Ross, 1964; Holubarsch et al. 1996). Overall, along with myocyte loss, energy metabolism disturbances, vasculature rearrangement, and other functional changes of failing myocytes (e.g. depressed Ca2+ handling, lower force output, and slower cross-bridge cycling), the redistribution of myocytes toward shallow length–tension relationships may contribute to depressed Frank–Starling relationships of failing hearts.
References Adhikari BB, Regnier M, Rivera AJ, Kreutziger KL & Martyn DA (2004). Cardiac length dependence of force and force redevelopment kinetics with altered cross-bridge cycling. Biophys J 87, 1784–1794. Ait mou Y, le Guennac J-Y, Mosca E, de Tombe PP & Cazorla O (2008). Differential contribution of cardiac sarcomeric proteins in the myofibrillar force response to stretch. Pflugers Arch 457, 25–36. Allen JD & Moss RL (1987). Factors influencing the ascending limb of the sarcomere length-tension relationship in rabbit skinned muscle fibers. J Physiol 390, 119–136. Bardswell SC & Kentish, J (2003). Resting and active sarcomere lengths in isolated rat ventricular myocytes. Biophys J 84, 431a. Braunwald E & Ross J (1964). Applicability of Starling’s law of the heart to man. Circ Res Suppl ii, 169–181. Brenner B (1988). Effect of calcium on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction. Proc Natl Acad Sci U S A 85, 3265–3269. Bristow M, Ginsburg R, Minobe W, Cubicciotti R, Sageman W, Billingham M, Harrison D & Stinson E (1982). Decrease catecholamine sensitivity and β-adrenergic-receptor density in failing human hearts. N Engl J Med 307, 205–211. Campbell K (1997). Rate constant of muscle force redevelopment reflects cooperative activation as well as cross-bridge kinetics. Biophys J 72, 254–262. Close RI (1972). The relations between sarcomere length and characteristics of isometric twitch contractions of frog sartorius muscle. J Physiol 220, 745–762. Drummond GB (2009). Reporting ethical matters in The Journal of Physiology: standards and advice. J Physiol 587, 713–719. Fabiato A (1988). Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol 157, 378–417. Fabiato A & Fabiato F (1975). Dependence of the contractile activation of skinned cardiac cells on the sarcomere length. Nature 256, 54–56. Fitzsimons DP, Patel JR, Campbell KS & Moss RL (2001a). Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers. J Gen Physiol 117, 133–148.
2902
L. M. Hanft and K. S. McDonald
Fitzsimons DP, Patel JR & Moss RL (2001b). Cross-bridge interaction kinetics in rat myocardium are accelerated by strong binding of myosin to the thin filament. J Physiol 530, 263–272. Fuchs F & Martyn DA (2005). Length-dependent Ca2+ activation in cardiac muscle: some remaining questions. J Muscle Res Cell Motil 26, 199–212. Gillis TE, Martyn DA, Rivera AJ & Regnier M (2007). Investigation of thin filament near-neighbor regulatory unit interactions during force development in skinned cardiac and skeletal muscle. J Physiol 580, 561–576. Gordon AM, Homsher E & Regnier M (2000). Regulation of contraction in striated muscle. Physiol Rev 80, 853–924. Gordon AM, Huxley AF & Julian FJ (1966). The variation in isometric tension with sarcomere length in vertebrate muscle fibers. J Physiol 184, 170–192. Guyton AC, Jones CE & Coleman TG (1973). Circulatory Physiology: Cardiac Output and its Regulation. W. B. Saunders Co. Philadelphia, PA. Hanft LM & McDonald KS (2009). Sarcomere length dependence of power output is increased after PKA treatment in rat cardiac myocytes. Am J Physiol Heart Circ Physiol 296, H1524–H1531. Hanft LM & McDonald KS (2010). Determinants of loaded shortening in cardiac myocytes. Biophys J (Abstract). Hinken AC & McDonald KS (2004). Inorganic phosphate speeds loaded shortening in rat skinned cardiac myocytes. Am J Physiol Cell Physiol 287, C500–C507. Holubarsch C, Ruf T, Goldstein DJ, Ashton RC, Nickl W, Pieske B, Pioch K, Ludemann J, Wiesner S, Hasenfuss G, Posival H, Just H & Burkhoff D (1996). Existence of the Frank-Starling mechanism in the failing human heart: investigations on the organ, tissue, and sarcomere levels. Circulation 94, 683–689. Kentish JC, McCloskey DT, Layland J, Palmer S, Leiden JM, Martin AF & Solaro RJ (2001). Phosphorylation of troponin I by protein kinase A accelerates relaxation and crossbridge cycle kinetics in mouse ventricular muscle. Circ Res 88, 1059–1065. Kentish JC & Stienen GJ (1994). Differential effects of length on maximal force production in myofibrillar ATPase activity in rat skinned cardiac muscle. J Physiol 475, 175–184. Kentish JC, ter Keurs HEDJ, Ricciardi L, Bucx JJJ & Noble MIM (1986). Comparison between the sarcomere length-force relations of intact and skinned trabeculae from rat right ventricle. Influence of calcium concentrations on these relations. Circ Res 58, 755–768. Konhilas JP, Irving TC & de Tombe PP (2002). Length-dependent activation in three striated muscle types of the rat. J Physiol 544, 225–236. Konhilas JP, Irving TC, Wolska BM, Jweied EE, Martin AF, Solaro RJ & deTombe PP (2003). Troponin I in the murine myocardium: influence on length-dependent activation and interfilament spacing. J Physiol 547, 951–961. Korte FS & McDonald KS (2007). Sarcomere length dependence of rat skinned cardiac myocyte mechanical properties: dependence on myosin heavy chain. J Physiol 581, 725–739.
J Physiol 588.15
Korte FS, McDonald KS, Harris SP & Moss RL (2003). Loaded shortening, power output, and rate of force redevelopment are increased with knockout of cardiac myosin binding protein-C. Circ Res 93, 752–758. Kreutziger KL, Pirroddi N, Scellini B, Tesi C, Poggesi C, Regnier M (2008). Thin filament calcium binding properties and regulatory unit interactions alter kinetics of tension development and relaxation in rabbit skeletal muscle. J Physiol 586, 3683–3700. Lakatta EG (1992). Length modulation of muscle performance: Frank-Starling Law of the Heart. In The Heart and Cardiovascular System, ed. Fozzard HA, pp. 1325–1351. Raven Press, New York. McDonald KS (2000). Ca2+ dependence of loaded shortening in rat skinned cardiac myocytes and skeletal muscle fibers. J Physiol 525, 169–181. McDonald KS, Wolff MR & Moss RL (1997). Sarcomere length dependence of the rate of tension redevelopment and submaximal tension in rat and rabbit skinned skeletal muscle fibers. J Physiol 501, 607–621. McDonald KS, Wolff MR & Moss RL (1998). Force-velocity and power-load curves in rat skinned cardiac myocytes. J Physiol 511, 519–531. Metzger JM, Greaser ML & Moss RL (1989). Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers: implications for twitch potentiation in intact muscle. J Gen Physiol 93, 855–883. Metzger JM & Moss RL (1990). Calcium-sensitive cross-bridge transitions in mammalian fast and slow skeletal muscle fibers. Science 247, 1088–1090. Moss RL (1979). Sarcomere length-tension relations of frog skinned muscle fibres during calcium activation at short lengths. J Physiol 292, 177–202. Moss RL, Nwoye LO & Greaser ML (1991). Substitution of cardiac troponin C into rabbit muscle does not alter length dependence of calcium sensitivity of tension. J Physiol 440, 273–289. Regnier M, Martin H, Barsotti RJ, Rivera AJ, Martyn DA & Clemmons E (2004). Cross-bridge versus thin filament contributions to the level and rate of force development in cardiac muscle. Biophys J 87, 1815–1824. Regnier MR, Rivera AJ, Chase PB, Smillie LB & Sorenson MM (1999). Regulation of skeletal muscle tension redevelopment by troponin C constructs with different calcium affinities. Biophys J 76, 2664–2672. Ross J & Braunwald E (1964). Studies on Starling’s law of the heart: the effect of impeding venous return on performance of the normal and failing human left ventricle. Circulation 30, 719–727. Solaro RJ (1986). Protein Phosphorylation in Heart Muscle. CRC Press, Boca Raton, FL, USA. Strang KT, Sweitzer NK, Greaser ML & Moss RL (1994). β-Adrenergic receptor stimulation increases unloaded shortening velocity of skinned single ventricular myocytes from rats. Circ Res 74, 542–549. Swartz DR & Moss RL (1992). Influence of a strong-binding myosin analog on Ca2+ sensitive mechanical properties of skinned skeletal muscle fibers. J Biol Chem 267, 20497–20506. C 2010 The Authors. Journal compilation C 2010 The Physiological Society
J Physiol 588.15
Cardiac myocyte length–tension relationships
Sweeney HL & Stull JT (1990). Alteration of cross-bridge kinetics by myosin light chain phosphorylation in rabbit skeletal muscle: implications for regulation of actin-myosin interaction. Proc Natl Acad Sci U S A 87, 414–418. Takimoto E, Soergel DG, Janssen PML, Stull LB, Kass DA & Murphy AM (2004). Frequency- and afterload-dependent cardiac modulation in vivo by troponin I with constitutively active protein kinase A phosphorylation sites. Circ Res 94, 496–504. Van Der Velden J, de Jong JW, Owen VJ, Burton PBJ & Stienen GJM (2000). Effect of protein kinase A on calcium sensitivity of force and its sarcomere length dependence in human cardiomyocytes. Cardiovasc Res 46, 487–495. Vannier C, Chevassus H & Vassort G (1996). Ca-dependence of isometric kinetics in single skinned ventricular cardiomyocytes from rats. Cardiovasc Res 32, 580–586. Wolff MR, McDonald KS & Moss RL (1995). Rate of tension development in cardiac muscle varies with level of activator calcium. Circ Res 76, 154–160.
C 2010 The Authors. Journal compilation C 2010 The Physiological Society
2903
Author contributions All experiments were performed in the laboratory of K.S.M. Both authors contributed equally in concept and design of experiments, collection, analysis and interpretation of data, and drafting and revising the article. Both authors approve the final version to be published.
Acknowledgements The authors are grateful to Dr Richard L. Moss and Dr F. Steven Korte for comments regarding earlier versions of this manuscript. This work was supported by a National Heart, Lung, and Blood Institute Grant (R01-HL-57852) to K.S.M. and an American Heart Association (Heartland Affiliate) Postdoctoral Fellowship (0825725G) to L.M.H.
