The Frank-Starling relationship of the heart yields increased stroke volume with greater end-diastolic volume, and this relationship is steeper after β-adrenergic stimulation. The underlying basis for the Frank-Starling mechanism involves length-dependent changes in both Ca2+ sensitivity of myofibrillar force and power output. In this study, we tested the hypothesis that PKA-induced phosphorylation of myofibrillar proteins would increase the length dependence of myofibrillar power output, which would provide a myofibrillar basis to, in part, explain the steeper Frank-Starling relations after β-adrenergic stimulation. For these experiments, adult rat left ventricles were mechanically disrupted, permeabilized cardiac myocyte preparations were attached between a force transducer and position motor, and the length dependence of loaded shortening and power output were measured before and after treatment with PKA. PKA increased the phosphorylation of myosin binding protein C and cardiac troponin I, as assessed by autoradiography. In terms of myocyte mechanics, PKA decreased the Ca2+ sensitivity of force and increased loaded shortening and power output at all relative loads when the myocyte preparations were at long sarcomere length (∼2.30 μm). PKA had less of an effect on loaded shortening and power output at short sarcomere length (∼2.0 μm). These changes resulted in a greater length dependence of myocyte power output after PKA treatment; peak normalized power output increased ∼20% with length before PKA and ∼40% after PKA. These results suggest that PKA-induced phosphorylation of myofibrillar proteins explains, in part, the steeper ventricular function curves (i.e., Frank-Starling relationship) after β-adrenergic stimulation of the left ventricle.
- protein kinase A
- myosin binding protein C
- cardiac troponin I
it has long been recognized that the relationship between end-diastolic volume and ventricular work (i.e., the Frank-Starling relationship) becomes steeper in response to β-adrenergic stimulation (35). Conversely, the Frank-Starling relationship becomes depressed with heart failure (19, 20, 36), and, interestingly, this coincides with downregulation of the β-adrenergic signaling system (2, 33, 34). It remains to be elucidated just how changes in β-adrenergic stimulation modify the Frank-Starling relationship. It is well established that β-adrenergic receptors are stimulated after the release of catecholamines from the sympathetic nervous system, and this leads to an enhanced contractile state of the myocardium (5, 8). Increased contractility is thought to be mediated by PKA, which phosphorylates several key regulatory proteins, including the sarcolemmal Ca2+ channel, phospholamban, cardiac troponin I (cTnI), and myosin binding protein C (MyBP-C) (37). Three of these phosphoproteins, the sarcolemmal Ca2+ channel, phospholamban, and cTnI, are intimately involved in the handling of myoplasmic Ca2+, suggesting that Ca2+ plays a prominent role in mediating the inotropic effects of β-adrenergic stimulation (5, 26). However, studies on both biochemical and mechanical properties of the isolated myocardium have suggested that β-adrenergic responses may also result from direct effects on cross-bridge cycling kinetics via PKA-induced phosphorylation of cTnI and the thick filament protein MyBP-C (14, 17, 22, 27, 39–42).
Understanding the effects that β-adrenergic stimulation has on the Frank-Starling relationship first necessitates an understanding of the factors that underlie the relationship. The mechanistic basis for the Frank-Starling relationship is thought to primarily involve length-dependent changes in the Ca2+ sensitivity of myofibrillar force and power output. The effect of PKA-induced phosphorylation of myofilaments on length dependence of Ca2+ sensitivity of force has yielded mixed results, whereby PKA has been reported to decrease (21), have minimal effect (43), and increase (23) length dependence of Ca2+ sensitivity of force in permeabilized mammalian cardiac muscle preparations. While these studies on isometric force production are important for addressing one mechanism by which PKA-induced phosphorylation modulates length-dependent cardiac myocyte function, it remains to be seen how PKA affects the length dependence of myocyte loaded shortening and power output, which ultimately dictates ventricular ejection during a cardiac cycle. In this study, we tested the hypothesis that PKA-induced phosphorylation of myofibrillar proteins would increase the length dependence of myofibrillar power output, which would provide a myofibrillar basis to, in part, explain the steeper Frank-Starling relations after β-adrenergic stimulation.
Male Sprague-Dawley rats (6 wk old) were obtained from Harlan (Madison, WI), housed in groups of two or three rats, and provided access to food and propranolol (1 mg/ml)-treated water ad libitum. Propranolol is a β-adrenergic antagonist and was administered in an attempt to normalize baseline PKA-induced phosphorylation levels of myofibrillar proteins in most animals. All procedures involving animal use were performed according to the Animal Care and Use Committee of the University of Missouri.
The compositions of relaxing and activating solutions were as follows (in mmol/l, obtained from Sigma at the highest possible purity): 1 free Mg2+, 7 EGTA, 4 MgATP, 20 imidazole, and 14.5 creatine phosphate (pH 7.0), with specific [Ca2+] between 10−4.5 (maximal Ca2+-activating solution) and 10−9 (relaxing solution) and sufficient KCl to adjust ionic strength to 180 mmol/l. Relaxing solution also contained 2 mM Pefabloc (Roche) to inhibit proteases and preserve the integrity of the myocytes.
Skinned cardiac myocytes were obtained by mechanical disruption of hearts from Sprague-Dawley rats as previously described (29). Briefly, rats (2–3 mo old) were anesthetized by the inhalation of isoflurane [20% (vol/vol) in olive oil], and their hearts were excised and rapidly placed in ice-cold relaxing solution. The left ventricle was separated from the right ventricle and dissected from the atria, cut into 2- to 3-mm pieces, and further disrupted for 5 s in a Waring blender. The resulting suspension of cells was centrifuged for 105 s at 165 g, after which the supernatant fluid was discarded. Myocytes were skinned by suspending the cell pellet for 5 min in 0.3% ultrapure Triton X-100 (Pierce Chemical) in cold relaxing solution. Skinned cells were washed twice with cold relaxing solution, suspended in 10 ml of relaxing solution, and kept on ice for the experimental day.
The experimental apparatus for the physiological measurements of myocyte preparations has been previously described (29). Briefly, a myocyte was attached between a force transducer and a high-speed motor by placing the ends of the myocyte into stainless steel troughs (25-gauge). The ends of the myocyte were secured by overlaying a 0.5-mm long piece of 3-0 monofilament nylon suture (Ethicon) onto each end of the myocyte and then tying the suture into the troughs with two loops of 10-0 monofilament suture (Ethicon).
Force-velocity and power-load measurements.
The protocol to obtain force-velocity and power-load measurements has been previously described in detail (31), and all measurements were done at 13 ± 1°C. To determine the effects of PKA on the SL dependence of loaded shortening and power output, the following protocol was used. An attached myocyte was adjusted to a SL of ∼2.30 μm, transferred into maximal Ca2+-activating solution (pCa 4.5), and then allowed to obtain maximal steady-state isometric force. It was transferred to a submaximal Ca2+-activating solution that yielded ∼0.60 maximal Ca2+-activated force at long SL (P4.5 long SL), and a series of subisometric force clamps were then applied to determine isotonic shortening velocities. The isotonic force was maintained using a servo system for 150–250 ms while muscle length changes during this time were monitored. Figure 1 shows a force clamp and the corresponding muscle length at long and short SL before and after PKA treatment. After the force clamp, the myocyte was slackened to near zero force to estimate the relative load sustained during the isotonic shortening, after which the myocyte was reextended to its starting length. Myocytes were kept in submaximal Ca2+-activating solution for 3–5 min, during which 15–25 force clamps were performed without a significant loss of force. If force fell below 80% of initial force, data from that myocyte were discarded. After force-velocity measurements at long SL, the SL was then shortened to ∼ 2.00 μm, and a second force-velocity relationship was obtained using the same submaximal Ca2+-activating solution. After these two series of force clamps, the myocyte was treated with the catalytic subunit of PKA (0.10 U/μl, Sigma) in relaxing solution for 45 min at 13 ± 1°C. After PKA treatment, the myocyte was activated with maximal Ca2+-activating solution, and a series of submaximal Ca2+ activations were then performed to determine the pCa solution that yielded ∼0.60 P4.5long SL. Force-velocity measurements were then obtained in this submaximal Ca2+-activating solution at both long and short SL.
Measurement of the rate of force redevelopment.
The kinetics of force redevelopment were obtained using a procedure previously described for skinned cardiac myocyte preparations (16, 25). While in Ca2+-activating solution, the myocyte preparation was rapidly shortened by ∼15% of the myocyte's initial length (Lo) to produce zero force. The myocyte preparation was then allowed to shorten for ∼20 ms; after 20 ms, the preparation was rapidly restretched to ∼105% of its Lo for 2 ms and then returned to Lo. The slack-restretch maneuver is thought to dissociate cross-bridges, and the subsequent force redevelopment is thought to arise from the reattachment of cross-bridges and transition to force-generating states. Force redevelopment measurements were carried out before the series of loaded contractions at both long and short SL and before and after PKA treatment. The protocol for force redevelopment measurements consisted of first setting the myocyte preparation to long SL and then performing a slack-restretch maneuver during maximal Ca2+ activation. A second rate of force redevelopment was measured during submaximal activation (∼60% P/P4.5 long SL) at long SL, which was made before the series of load clamps. The myocyte preparation was then adjusted to short SL, and a third force redevelopment trace was obtained (before load clamps at short SL). This protocol was repeated after the myocyte was incubated in PKA to obtain three more force redevelopment measurements (i.e., at long SL during maximal Ca2+ activation, at long SL during submaximal Ca2+ activation, and at short SL during submaximal Ca2+). To assure that PKA-induced changes in force redevelopment rates were not a result of preparation rundown, control experiments were performed in which myocyte preparations were incubated in PKA in the presence of protein kinase inhibitor (PKI; 300 μg/ml). To further assess whether rundown was a factor in PKA-induced changes in force redevelopment rates, a series of experiments were performed where only two slack-restretch maneuvers were performed: one during maximal Ca2+ activation at long SL before PKA and the second after a 45-min PKA incubation during maximal Ca2+ activation at long SL. For these measurements, Ca2+-activated force was the same or oftentimes higher after the PKA incubation, which indicated negligible mechanical rundown.
SDS-PAGE and autoradiography.
To determine the myofibrillar substrates of PKA, 100 μg of skinned cardiac myocytes were incubated with the catalytic subunit of PKA (0.1 U/μl) and 50 μCi [γ-32P]ATP for 45 min. The reaction was stopped by the addition of electrophoresis sample buffer and heating at 95°C for 3 min. Samples were then separated by SDS-PAGE, silver stained, dried, and subsequently exposed to X-ray film for ∼1 h at −70°C (Fig. 2).
Myocyte length traces, force-velocity curves, and power-load curves were analyzed as previously described (29). Briefly, myocyte length traces during loaded shortening were fit to the following single decaying exponential equation: (1) where L is cell length at time t, A and C are constants with dimensions of length, and k is the rate constant of shortening. The velocity of shortening at any given time t was determined as the slope of the tangent to the fitted curve at that time point. In this study, velocities of shortening were calculated by extrapolation of the fitted curve to the onset of the force clamp (i.e., t = 0).
Hyperbolic force-velocity curves were fit to the relative force-velocity data using the following Hill equation (15): (2) where P is force during shortening at velocity V, Po is the peak isometric force, and a and b are constants with dimensions of force and velocity, respectively. Power-load curves were obtained by multiplying force × velocity at each load on the force-velocity curve.
Curve fitting was performed using a customized program written in Qbasic as well as with commercial software (SigmaPlot).
Tension redevelopment after a slack-restretch maneuver was fit by the following single exponential equation: (3) where F is tension at time t, Fmax is maximal tension, and ktr is the rate constant of force development.
To determine if SL significantly affected loaded shortening velocity, power, or ktr, one-way repeated-measures tests were used, and a Student-Neuman-Keuls test was performed as a post hoc test to assess differences between group means. For force redevelopment experiments that consisted of just two measurements (one at long SL before PKA and the other at long SL after PKA), a paired t-test was performed. In all cases, P < 0.05 was accepted as significant. Data are presented as means ± SD; n is the number of myocyte preparations.
Effect of PKA on protein phosphorylation and Ca2+ sensitivity of force.
Treatment of permeabilized cardiac myocyte preparations with PKA caused increased phosphorylation of MyBP-C and cTnI (Fig. 2) and decreased the Ca2+ sensitivity of isometric force. After PKA treatment, greater activator Ca2+ was needed to produce similar submaximal isometric force [expressed as force produced at long sarcomere length with pCa 4.5 solution (P/P4.5 long SL)]. For instance, before PKA treatment, pCa 5.73 ± 0.14 produced 0.59 ± 0.08 P/P4.5 long SL at long SL and 0.25 ± 0.05 P/P4.5 long SL at short SL. After PKA treatment, pCa 5.57 ± 0.10 was needed to produce 0.57 ± 0.10 P/P4.5 long SL at long SL and 0.25 ± 0.06 P/P4.5 long SL at short SL. The observed decrease in Ca2+ sensitivity of force is consistent with numerous reports in the literature on cardiac muscle preparations (14, 18, 21, 23, 39, 41, 43, 46, 47).
Effect of PKA on SL dependence of loaded shortening and power output.
The effect of PKA on the SL dependence of loaded shortening was examined by measuring force-velocity relationships at long (∼2.30 μm) and short (∼2.0 μm) SL before and after PKA treatment of cardiac myocyte preparations. Before PKA treatment, a reduction in SL resulted in a decrease in isometric force (at the same activator [Ca2+]) and a downward shift in the force-velocity relationship (Fig. 1C) [similar to our previous report (24) using euthyroid rat cardiac myocyte preparations]. After PKA treatment, myocyte loaded shortening velocity was sped at virtually all loads at the longer SL, resulting in a ∼30% increase in peak normalized power output (Fig. 1D) (before PKA peak normalized power output = 0.199 ± 0.080 P/Po·μm·s−1·sarcomere−1 and after PKA peak normalized power output = 0.263 ± 0.098 P/Po·μm·s−1·sarcomere−1). This result is quantitatively similar to one previously reported (14). PKA treatment had less of an effect on force-velocity and power-load curves at short SL. At short SL, peak normalized power output only increased ∼10% after PKA treatment (before PKA peak normalized power output = 0.169 ± 0.078 P/Po·μm·s−1·sarcomere−1 and after PKA peak normalized power output = 0.185 ± 0.076 P/Po·μm·s−1·sarcomere−1). Thus, the length dependence of loaded shortening and power output was increased after PKA treatment due, in large part, to the effect of PKA at long SL. Overall, peak normalized power output increased ∼40% with increased SL after PKA (0.185 ± 0.076 P/Po·μm·s−1·sarcomere−1 at short SL vs. 0.263 ± 0.098 P/Po·μm·s−1·sarcomere−1 at long SL), whereas without PKA treatment, peak normalized power increased ∼20% with increased SL (short SL = 0.169 ± 0.078 P/Po·μm·s−1·sarcomere−1 and long SL = 0.199 ± 0.080 P/Po·μm·s−1·sarcomere−1). Interestingly, when peak power generating capacity was expressed in absolute values, peak power increased ∼3.5-fold with increased SL after PKA treatment (long SL = 2.88 ± 1.83 μW/mg compared with short SL = 0.83 ± 0.58 μW/mg). In contrast, untreated myocytes showed only an ∼2.3-fold increase in peak absolute power with increased SL, going from 0.87 ± 0.72 μW/mg at short SL to 2.01 ± 1.33 μW/mg at long SL. Overall, these results indicate a greater SL dependence of loaded shortening and power output after PKA-induced phosphorylation of myofibrillar proteins.
Effect of PKA on SL dependence of rates of force development.
Rates of force development were measured in cardiac myocyte preparations at long and short SL before and after PKA treatment. Before PKA (and during submaximal Ca2+ activations), a decrease in SL had no effect on ktr (Fig. 3B) despite a significant decrease in isometric force. This result is consistent with previous reports (1, 24, 38) that made similar mechanical measurements. After PKA treatment, there was a decrease in ktr at both long SL (see force traces in Fig. 3) and short SL during submaximal Ca2+ activations. This result is in contrast to previous reports (7, 39) that found that PKA treatment increased ktr in mouse myocardial preparations. The decline in ktr after PKA was similar at both long and short SL, which yielded no change in the SL dependence of force development rates after PKA treatment. Interestingly, PKA not only decreased ktr but also, during submaximal Ca2+ activations, yielded a marked increase in the transient force overshoot after the slack-restretch maneuver used to induce force redevelopment (Fig. 3C). Control experiments were performed by treating myocytes with PKA in the presence of PKI. This treatment prevented both the slowdown in force redevelopment and the increase in transient force overshoot that was observed with PKA alone (data not shown). Additional measurements examined the effects of PKA on the rates of force redevelopment before and after PKA during maximal Ca2+ activations. Some of these measurements were made in the same preparations that loaded shortening was measured, whereas others were done on preparations that just incorporated as few as two slack-restretch maneuvers (one before PKA and one after PKA to help avoid any potential preparation rundown). In all cases, PKA treatment resulted in slower rates of force redevelopment during maximal Ca2+ activations (before PKA ktr = 5.26 ± 1.70 s−1 vs. after PKA ktr = 4.11 ± 1.75 s−1, P = 0.008, n = 17), even though force was unchanged (before PKA force = 13.8 ± 6.9 μN vs. after PKA force = 14.6 ± 6.8 μN, P = 0.122, n = 17).
PKA-induced phosphorylation of myofibrillar proteins yielded a greater length dependence of power output in rat skinned cardiac myocytes. This finding provides a myofibrillar mechanism to explain, in part, the long-standing observation that the Frank-Starling relationship becomes steeper in response to β-adrenergic stimulation. Experiments on intact hearts in vivo clearly demonstrated that rapidly acquired cardiac function curves were shifted upward and became steeper in response to sympathetic stimulation (12, 35). For instance, in young adult human hearts, the plateau of a cardiac function curve rises from ∼10 l/min during normal, basal sympathetic stimulation (at a right atrial pressure of ∼4 mmHg) up to ∼20–25 l/min during maximal sympathetic stimulation, such as occurs during heavy exercise loads (11). As a consequence of this increased contractility, the Frank-Starling relationship becomes much steeper during elevated sympathetic stimulation over a right atrial pressure range of approximately −1 to 5 mmHg. One plausible mechanism that may account for the steeper Frank-Starling relationship is a greater length dependence of Ca2+ sensitivity of force, which is thought to be the primary cause of increased stroke volume with increased end-diastolic volume (9). However, studies investigating the effects of PKA-induced phosphorylation of myofibrillar proteins on the length dependence of Ca2+ sensitivity of force have yielded mixed results. Kajiwara et al. (21) reported that PKA actually decreased the SL dependence of Ca2+ sensitivity of force in permeabilized rat cardiac trabecular preparations. van der Velden et al. (43) reported that PKA had minimal effects on the SL dependence of Ca2+ sensitivity of force in human permeabilized cardiac myocyte preparations, although there was a trend for a greater length dependence of isometric force after PKA in failing human heart myocyte preparations when values were expressed as ΔEC50. Konhilas et al. (23) reported that PKA enhanced the length dependence of Ca2+ sensitivity of force in mouse permeabilized cardiac myocyte preparations when data were expressed as ΔEC50. This effect seemed to be due to the phosphorylation of cTnI since PKA did not alter ΔEC50 in transgenic mice that contained slow skeletal troponin I (ssTnI), which lacks PKA phosphorylation sites. Another possible mechanism to explain the steeper Frank-Starling relationships is a greater length dependence of loaded shortening after sympathetic stimulation. Since stroke volume is ultimately determined by the amount of myocyte shortening during ejection, if the degree of loaded shortening increases with changes in SL after PKA-induced phosphorylation, this could explain steeper ventricular function curves in response to sympathetic stimulation. To test this idea, we directly measured the SL dependence of loaded shortening (and power output) before and after treatment of cardiac myocyte preparations with PKA. We found that peak normalized power output increased ∼20% with increased SL before PKA treatment, and this was elevated to an ∼40% increase after PKA treatment. The greater length dependence of loaded shortening and power output after PKA treatment of rat myocyte preparations resulted primarily from an upward shift in the force-velocity relationship at long SL, as PKA had only a minimal effect on loaded shortening at short SL.
Another important factor in determining stroke volume is the number of force-generating cross-bridges produced during the isovolumic contraction phase of the cardiac cycle. This is important in two regards: first, the rate of force-generating transitions dictates the rate of ventricular pressure development, which determines the systolic time spent during the isovolumic contraction phase. The less time that the ventricle spends in this phase, the more systolic time there is for ejection. Second, the number of force-generating cross-bridges formed during isovolumic contraction affects the load per cross-bridge and, ultimately, the rate that cross-bridges cycle and the ventricle shortens during ejection. We found that the rate of force development was altered by PKA in rat cardiac myocyte preparations. Interestingly, PKA caused two mechanical consequences of force redevelopment: 1) rates of force redevelopment were slowed at both long and short SL and 2) there was a marked increase in the transient overshoot of force developed after the mechanical perturbation. In one sense, this would seem to reduce ejection after PKA by increasing the time needed for isovolumic contraction. On the other hand, the large tension overshoot implies greater stretch activation after PKA, which is consistent with previous reports (39, 40). More stretch activation would tend to increase the number of cross-bridges to work against the afterload, thereby augmenting loaded shortening and enhancement of ejection. Just how PKA-induced phosphorylation augments stretch activation (e.g., more tension overshoot after a slack-restretch) is unclear but may involve changes in the stiffness of the thin filaments. We observed two additional conditions that reduced the amount of tension overshoot in skinned striated muscle preparations: 1) a reduction in SL (30) and 2) partial cleavage of titin (using 25 ng/ml trypsin; unpublished observations). Both of these conditions would most likely reduce thin filament stiffness (due to a more slackened titin) and perhaps reduce the mechanical signals involved in the transmission of cooperative activation along the thin filaments. Following this line of reasoning, a stiffer thin filament would help transmit cooperative activation signals induced by Ca2+ binding to troponin and/or cross-bridge binding. This idea is consistent with a spatial mechanical model of the half-sarcomere, which computed greater cooperative activation with stiffer thin filaments (4). Whether PKA-induced phosphorylation actually alters thin filament stiffness has not been tested but is not consistent with the fact that PKA phosphorylation tends to reduce myocyte stiffness; however, this effect may be mostly related to titin per se (10). Also, a recent sarcomere model yielded a greater tension overshoot with more compliant thick and thin filaments rather than stiffer filaments (3).
Interestingly, we observed that PKA treatment decreased ktr during both maximal and submaximal Ca2+ activations in rat cardiac myocyte preparations. This result contrasts with previous reports (7, 39) that found that PKA treatment increased ktr in mouse myocardial preparations. The reason(s) for this difference is unclear but does not appear to be a species-specific phenomenon as we also observed a slowdown in force redevelopment with PKA in mouse myocyte preparations. It is possible that the opposing results arise from inherent differences in the preparations.
So just how does PKA-induced phosphorylation increase the SL dependence of loaded shortening and power output? One plausible idea is that PKA-induced phosphorylation of MyBP-C loosens its constraint on the thick filament and yields more flexible cross-bridges. This could cause faster loaded shortening by faster cycling kinetics per se or faster cycle kinetics due to more bound cross-bridges resulting from the greater cooperative activation of the thin filament. This idea is based on three previous findings: 1) genetic ablation of MyBP-C yielded faster loaded shortening velocities (25); 2) PKA-induced phosphorylation yielded similar stretch activation properties as ablation of MyBP-C (39, 40); and 3) short SL yielded faster loaded shortening when Ca2+-activated force was matched to long SL (24).
In each case, it is conceivable that MyBP-C's constraint on cross-bridges is loosened either by ablation of MyBP-C, PKA-induced phosphorylation of MyBP-C, or due to a more slackened titin (24). However, experimental evidence has not ruled out that phosphorylation of cTnI is necessary to increase the SL dependence of loaded shortening. This idea needs to be tested by replacement of cTnI that lacks PKA phosphorylation sites (i.e., Ser23/24). It is also possible that PKA-induced phosphorylation of titin is necessary to elicit a greater SL dependence of loaded shortening. PKA-induced phosphorylation of titin has been shown to reduce passive force in cardiac myocytes (10); this result is consistent with the concept of a mechanical signal that loosens MyBP-C's constraint on cross-bridges.
The result that PKA induced-phosphorylation of myofilaments caused greater length dependence of loaded shortening and power output likely has important physiological and perhaps pathophysiological relevance. First, it provides an important myofibrillar mechanism to, at least in part, explain the increased cardiac reserve in response to sympathetic stimulation. This underlying basis for a steeper Frank-Starling relationship provides a means for healthy ventricles to adjust output to meet peripheral demands on a beat-to-beat basis. Interestingly, a recent study (13) found that β-adrenergic blockade of highly trained subjects markedly attenuated the increase in stroke volume normally associated with exercise. A likely explanation for this is that inhibition of the normal shift in ventricular function curves associated with sympathetic (β-adrenergic) stimulation and its consequences of greater power at the myofibrillar level. From a pathophysiological standpoint, heart failure is defined as the inability of the ventricular output to meet peripheral energy demands and is characterized by a downward and rightward shift of ventricular function curves (i.e., depressed Frank-Starling relationship). A downward shift in ventricular function curves is also characteristic of reduced sympathetic stimulation, and several studies (2, 33, 34) on the human myocardium from failing hearts have shown downregulation of the β-adrenergic signaling system and depressed PKA-induced phosphorylation of myofibrillar proteins (28, 32, 43–45, 48, 49). Together with the results of this study, these finding implicate attenuation of the length dependence of loaded shortening with decreased PKA phosphorylation of myofilaments as potentially causative in the depressed Frank-Starling relationship in failing hearts.
This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-57852 and K02-HL-71550 (to K. S. McDonald) and National Arthritis and Musculoskeletal and Skin Diseases Grant T32-AR-048523 and American Heart Association (Heartland Affiliate) Postdoctoral Fellowship 0825725G (to L. M. Hanft).
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