Julian E. Stelzer

Differential Roles of Cardiac Myosin-Binding Protein C and Cardiac Troponin I in the Myofibrillar Force Responses to Protein Kinase A Phosphorylation

The heart is remarkably adaptable in its ability to vary its function to meet the changing demands of the circulatory system. During times of physiological stress, cardiac output increases in response to increased sympathetic activity, which results in protein kinase A (PKA)-mediated phosphorylations of the myofilament proteins cardiac troponin (cTn)I and cardiac myosin-binding protein (cMyBP)-C. Despite the importance of this mechanism, little is known about the relative contributions of cTnI and cMyBP-C phosphorylation to increased cardiac contractility. Using engineered mouse lines either lacking cMyBP-C (cMyBP-C−/−) or expressing a non-PKA phosphorylatable cTnI (cTnIala2), or both (cMyBP-C−/−/cTnIala2), we investigated the roles of cTnI and cMyBP-C phosphorylation in the regulation of the stretch-activation response. PKA treatment of wild-type and cTnIala2 skinned ventricular myocardium accelerated stretch activation such that the response was indistinguishable from stretch activation of cMyBP-C−/− or cMyBP-C−/−/cTnIala2 myocardium; however, PKA had no effect on stretch activation in cMyBP-C−/− or cMyBP-C−/−/cTnIala2 myocardium. These results indicate that the acceleration of stretch activation in wild-type and cTnIala2 myocardium is caused by phosphorylation of cMyBP-C and not cTnI. We conclude that the primary effect of PKA phosphorylation of cTnI is reduced Ca2+ sensitivity of force, whereas phosphorylation of cMyBP-C accelerates the kinetics of force development. These results predict that PKA phosphorylation of myofibrillar proteins in living myocardium contributes to accelerated relaxation in diastole and increased rates of force development in systole.

Acceleration of Crossbridge Kinetics by Protein Kinase A Phosphorylation of Cardiac Myosin Binding Protein C Modulates Cardiac Function

Normal cardiac function requires dynamic modulation of contraction. β1-Adrenergic–induced protein kinase (PK)A phosphorylation of cardiac myosin binding protein (cMyBP)-C may regulate crossbridge kinetics to modulate contraction. We tested this idea with mechanical measurements and echocardiography in a mouse model lacking 3 PKA sites on cMyBP-C, ie, cMyBP-C(t3SA). We developed the model by transgenic expression of mutant cMyBP-C with Ser-to-Ala mutations on the cMyBP-C knockout background. Western blots, immunofluorescence, and in vitro phosphorylation combined to show that non–PKA-phosphorylatable cMyBP-C expressed at 74% compared to normal wild-type (WT) and was correctly positioned in the sarcomeres. Similar expression of WT cMyBP-C at 72% served as control, ie, cMyBP-C(tWT). Skinned myocardium responded to stretch with an immediate increase in force, followed by a transient relaxation of force and finally a delayed development of force, ie, stretch activation. The rate constants of relaxation, krel (s-1), and delayed force development, kdf (s-1), in the stretch activation response are indicators of crossbridge cycling kinetics. cMyBP-C(t3SA) myocardium had baseline krel and kdf similar to WT myocardium, but, unlike WT, krel and kdf were not accelerated by PKA treatment. Reduced dobutamine augmentation of systolic function in cMyBP-C(t3SA) hearts during echocardiography corroborated the stretch activation findings. Furthermore, cMyBP-C(t3SA) hearts exhibited basal echocardiographic findings of systolic dysfunction, diastolic dysfunction, and hypertrophy. Conversely, cMyBP-C(tWT) hearts performed similar to WT. Thus, PKA phosphorylation of cMyBP-C accelerates crossbridge kinetics and loss of this regulation leads to cardiac dysfunction.

Protein Kinase A-Mediated Acceleration of the Stretch Activation Response in Murine Skinned Myocardium Is Eliminated by Ablation of cMyBP-C

&bgr;-Adrenergic agonists induce protein kinase A (PKA) phosphorylation of the cardiac myofilament proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI), resulting in enhanced systolic function, but the relative contributions of cMyBP-C and cTnI to augmented contractility are not known. To investigate possible roles of cMyBP-C in this response, we examined the effects of PKA treatment on the rate of force redevelopment and the stretch activation response in skinned ventricular myocardium from both wild-type (WT) and cMyBP-C null (cMyBP-C−/−) myocardium. In WT myocardium, PKA treatment accelerated the rate of force redevelopment and the stretch activation response, resulting in a shorter time to the peak of delayed force development when the muscle was stretched to a new isometric length. Ablation of cMyBP-C accelerated the rate of force redevelopment and stretch activation response to a degree similar to that observed in PKA treatment of WT myocardium; however, PKA treatment had no effect on the rate of force development and the stretch activation response in null myocardium. These results indicate that ablation of cMyBP-C and PKA treatment of WT myocardium have similar effects on cross-bridge cycling kinetics and suggest that PKA phosphorylation of cMyBP-C accelerates the rate of force generation and thereby contributes to the accelerated twitch kinetics observed in living myocardium during &bgr;-adrenergic stimulation.

Ablation of Cardiac Myosin-Binding Protein-C Accelerates Stretch Activation in Murine Skinned Myocardium

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament accessory protein that binds tightly to myosin, but despite evidence that mutations in the cMyBP-C gene comprise a frequent cause of hypertrophic cardiomyopathy, relatively little is known about the role(s) of cMyBP-C in myocardium. Based on earlier studies demonstrating the potential importance of stretch activation in cardiac contraction, we examined the effects of cMyBP-C on the stretch activation responses of skinned ventricular preparations from wild-type (WT) and homozygous cMyBP-C knockout mice (cMyBP-C−/−) previously developed in our laboratory. Sudden stretch of skinned myocardium during maximal or submaximal Ca2+ activations resulted in an instantaneous increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force (ie, stretch activation) to levels greater than prestretch force. Ablation of cMyBP-C dramatically altered the stretch activation response, ie, the rates of force decay and delayed force transient were accelerated compared with WT myocardium. These results suggest that cMyBP-C normally constrains the spatial position of myosin cross-bridges, which, in turn, limits both the rate and extent of interaction of cross-bridges with actin. We propose that ablation of cMyBP-C removes this constraint, increases the likelihood of cross-bridge binding to actin, and speeds the rate of delayed force development following stretch. Regardless of the specific mechanism, acceleration of cross-bridge cycling in cMyBP-C−/− myocardium could account for the abbreviation of systolic ejection in this mouse as a direct consequence of premature stretch activation of ventricular myocardium.

Acceleration of Stretch Activation in Murine Myocardium due to Phosphorylation of Myosin Regulatory Light Chain

The regulatory light chains (RLCs) of vertebrate muscle myosins bind to the neck region of the heavy chain domain and are thought to play important structural roles in force transmission between the cross-bridge head and thick filament backbone. In vertebrate striated muscles, the RLCs are reversibly phosphorylated by a specific myosin light chain kinase (MLCK), and while phosphorylation has been shown to accelerate the kinetics of force development in skeletal muscle, the effects of RLC phosphorylation in cardiac muscle are not well understood. Here, we assessed the effects of RLC phosphorylation on force, and the kinetics of force development in myocardium was isolated in the presence of 2,3-butanedione monoxime (BDM) to dephosphorylate RLC, subsequently skinned, and then treated with MLCK to phosphorylate RLC. Since RLC phosphorylation may be an important determinant of stretch activation in myocardium, we recorded the force responses of skinned myocardium to sudden stretches of 1% of muscle length both before and after treatment with MLCK. MLCK increased RLC phosphorylation, increased the Ca2+ sensitivity of isometric force, reduced the steepness of the force–pCa relationship, and increased both Ca2+-activated and Ca2+-independent force. Sudden stretch of myocardium during an otherwise isometric contraction resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force, i.e., stretch activation, to levels greater than pre-stretch force. MLCK had profound effects on the stretch activation responses during maximal and submaximal activations: the amplitude and rate of force decay after stretch were significantly reduced, and the rate of delayed force recovery was accelerated and its amplitude reduced. These data show that RLC phosphorylation increases force and the rate of cross-bridge recruitment in murine myocardium, which would increase power generation in vivo and thereby enhance systolic function.

Role of myosin heavy chain composition in the stretch activation response of rat myocardium

The speed and force of myocardial contraction during systolic ejection is largely dependent on the intrinsic contractile properties of cardiac myocytes. As the myosin heavy chain (MHC) isoform of cardiac muscle is an important determinant of the contractile properties of individual myocytes, we studied the effects of altered MHC isoform expression in rat myocardium on the mechanical properties of skinned ventricular preparations. Skinned myocardium from thyroidectomized rats expressing only the β MHC isoform displayed rates of force redevelopment that were about 2.5‐fold slower than in myocardium from hyperthyroid rats expressing only the α MHC isoform, but the amount of force generated at a given level of Ca2+ activation was not different. Because recent studies suggest that the stretch activation response in myocardium has an important role in systolic function, we also examined the effect of MHC isoform expression on the stretch activation response by applying a rapid stretch (1% of muscle length) to an otherwise isometrically contracting muscle fibre. Sudden stretch of myocardium resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force (i.e. stretch activation) to levels greater than prestretch force. β MHC expression dramatically slowed the overall rate of the stretch activation response compared to expression of α MHC isoform; specifically, the rate of force decay was ∼2‐fold slower and the rate of delayed force development was ∼2.5‐fold slower. In contrast, MHC isoform had no effect on the amplitude of the stretch activation response. Collectively, these data show that expression of β MHC in myocardium dramatically slows rates of cross‐bridge recruitment and detachment which would be expected to decrease power output and contribute to depressed systolic function in end‐stage heart failure.

Contributions of Stretch Activation to Length-dependent Contraction in Murine Myocardium

The steep relationship between systolic force production and end diastolic volume (Frank-Starling relationship) in myocardium is a potentially important mechanism by which the work capacity of the heart varies on a beat-to-beat basis, but the molecular basis for the effects of myocardial fiber length on cardiac work are still not well understood. Recent studies have suggested that an intrinsic property of myocardium, stretch activation, contributes to force generation during systolic ejection in myocardium. To examine the role of stretch activation in length dependence of activation we recorded the force responses of murine skinned myocardium to sudden stretches of 1% of muscle length at both short (1.90 μm) and long (2.25 μm) sarcomere lengths (SL). Maximal Ca2+-activated force and Ca2+ sensitivity of force were greater at longer SL, such that more force was produced at a given Ca2+ concentration. Sudden stretch of myocardium during an otherwise isometric contraction resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed development of force, i.e., stretch activation, to levels greater than prestretch force. At both maximal and submaximal activations, increased SL significantly reduced the initial rate of force decay following stretch; at submaximal activations (but not at maximal) the rate of delayed force development was accelerated. This combination of mechanical effects of increased SL would be expected to increase force generation during systolic ejection in vivo and prolong the period of ejection. These results suggest that sarcomere length dependence of stretch activation contributes to the steepness of the Frank-Starling relationship in living myocardium.

Transmural variation in myosin heavy chain isoform expression modulates the timing of myocardial force generation in porcine left ventricle

Recent studies have shown that the sequence and timing of mechanical activation of myocardium vary across the ventricular wall. However, the contributions of variable expression of myofilament protein isoforms in mediating the timing of myocardial activation in ventricular systole are not well understood. To assess the functional consequences of transmural differences in myofilament protein expression, we studied the dynamic mechanical properties of multicellular skinned preparations isolated from the sub‐endocardial and sub‐epicardial regions of the porcine ventricular midwall. Compared to endocardial fibres, epicardial fibres exhibited significantly faster rates of stretch activation and force redevelopment (ktr), although the amount of force produced at a given [Ca2+] was not significantly different. Consistent with these results, SDS‐PAGE analysis revealed significantly elevated expression of α myosin heavy chain (MHC) isoform in epicardial fibres (13 ± 1%) versus endocardial fibres (3 ± 1%). Linear regression analysis revealed that the apparent rates of delayed force development and force decay following stretch correlated with MHC isoform expression (r2= 0.80 and r2= 0.73, respectively, P < 0.05). No differences in the relative abundance or phosphorylation status of other myofilament proteins were detected. These data show that transmural differences in MHC isoform expression contribute to regional differences in dynamic mechanical function of porcine left ventricles, which in turn modulate the timing of force generation across the ventricular wall and work production during systole.

Determination of rate constants for turnover of myosin isoforms in rat myocardium: Implications for in vivo contractile kinetics

The ventricles of small mammals express mostly alpha-myosin heavy chain (alpha-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express approximately 10% alpha-MHC on a predominately beta-MHC (slow isoform) background. In failing human ventricles, the amount of alpha-MHC is dramatically reduced, leading to the hypothesis that even small amounts of alpha-MHC on a predominately beta-MHC background confer significantly higher rates of force development in healthy ventricles. To test this hypothesis, it is necessary to determine the fundamental rate constants of cross-bridge attachment (f(app)) and detachment (g(app)) for myosins composed of 100% alpha-MHC or beta-MHC, which can then be used to calculate twitch time courses for muscles expressing variable ratios of MHC isoforms. In the present study, rat skinned trabeculae expressing either 100% alpha-MHC or 100% beta-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment (k(tr)) in solutions of varying Ca(2+) concentrations. The rate of ATP utilization was approximately 2.5-fold higher in preparations expressing 100% alpha-MHC compared with those expressing only beta-MHC, whereas k(tr) was 2-fold faster in the alpha-MHC myocardium. From these variables, we calculated f(app) to be approximately threefold higher for alpha-MHC than beta-MHC and g(app) to be twofold higher in alpha-MHC. Mathematical modeling of isometric twitches predicted that small increases in alpha-MHC significantly increased the rate of force development. These results suggest that low-level expression of alpha-MHC has significant effects on contraction kinetics.

Impaired contractile function due to decreased cardiac myosin binding protein C content in the sarcomere

Mutations in cardiac myosin binding protein C (MyBP-C) are a common cause of familial hypertrophic cardiomyopathy (FHC). The majority of MyBP-C mutations are expected to reduce MyBP-C expression; however, the consequences of MyBP-C deficiency on the regulation of myofilament function, Ca²⁺ homeostasis, and in vivo cardiac function are unknown. To elucidate the effects of decreased MyBP-C expression on cardiac function, we employed MyBP-C heterozygous null (MyBP-C+/-) mice presenting decreases in MyBP-C expression (32%) similar to those of FHC patients carrying MyBP-C mutations. The levels of MyBP-C phosphorylation were reduced 53% in MyBP-C+/- hearts compared with wild-type hearts. Skinned myocardium isolated from MyBP-C+/- hearts displayed decreased cross-bridge stiffness at half-maximal Ca²⁺ activations, increased steady-state force generation, and accelerated rates of cross-bridge recruitment at low Ca²⁺ activations (<15% and <25% of maximum, respectively). Protein kinase A treatment abolished basal differences in rates of cross-bridge recruitment between MyBP-C+/- and wild-type myocardium. Intact ventricular myocytes from MyBP-C+/- hearts displayed abnormal sarcomere shortening but unchanged Ca²⁺ transient kinetics. Despite a lack of left ventricular hypertrophy, MyBP-C+/- hearts exhibited elevated end-diastolic pressure and decreased peak rate of LV pressure rise, which was normalized following dobutamine infusion. Furthermore, electrocardiogram recordings in conscious MyBP-C+/- mice revealed prolonged QRS and QT intervals, which are known risk factors for cardiac arrhythmia. Collectively, our data show that reduced MyBP-C expression and phosphorylation in the sarcomere result in myofilament dysfunction, contributing to contractile dysfunction that precedes compensatory adaptations in Ca²⁺ handling, and chamber remodeling. Perturbations in mechanical and electrical activity in MyBP-C+/- mice could increase their susceptibility to cardiac dysfunction and arrhythmia.