Jitandrakumar R. Patel

Impaired cardiomyocyte relaxation and diastolic function in transgenic mice expressing slow skeletal troponin I in the heart

1 To assess the specific functions of the cardiac isoform of troponin I (cTnI), we produced transgenic mice that expressed slow skeletal troponin I (ssTnI) specifically in cardiomyocytes. Cardiomyocytes from these mice displayed quantitative replacement of cTnI with transgene‐encoded ssTnI. 2 The ssTnI transgenic mice were viable and fertile and did not display increased mortality or detectable cardiovascular histopathology. They exhibited normal ventricular weights and heart rates. 3 Permeabilized transgenic cardiomyocytes demonstrated an increased Ca2+ sensitivity of tension and a lack of contractile responsiveness to cAMP‐dependent protein kinase (PKA). Isolated cardiomyocytes from transgenic mice had normal velocities of unloaded shortening but unlike wild‐type controls exhibited no enhancement of the velocity of shortening in response to treatment with isoprenaline. Transgenic cardiomyocytes exhibited greater extents of shortening than non‐transgenic cardiomyocytes at baseline and after treatment with isoprenaline. 4 The rates of rise of intracellular [Ca2+] and the peak amplitudes of the intracellular [Ca2+] transients were similar in transgenic and wild‐type myocytes. However, the half‐time of intracellular [Ca2+] decay was significantly greater in the transgenic myocytes. This change in decay of intracellular [Ca2+] was correlated with an increase in the re‐lengthening time of the transgenic cells. 5 These changes in cardiomyocyte function in vitro were manifested in vivo as impaired diastolic function both at baseline and after stimulation with isoprenaline. 6 Thus, cTnI has important roles in regulating the Ca2+ sensitivity of cardiac myofibrils and controlling cardiomyocyte relaxation and cardiac diastolic function. cTnI is also required for the normal responsiveness of cardiomyocytes to β‐adrenergic receptor stimulation.

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.

Primary structure and properties of helothermine, a peptide toxin that blocks ryanodine receptors

Helothermine, a protein from the venom of the Mexican beaded lizard (Heloderma horridum horridum), was found to inhibit [3H]ryanodine binding to cardiac and skeletal sarcoplasmic reticulum, to block cardiac and skeletal ryanodine receptor channels incorporated into planar bilayers, and to block Ca(2+)-induced Ca2+ release triggered by photolysis of nitr-5 in saponin-permeabilized trabeculae from rat ventricle. Cloning of the helothermine cDNA revealed that the protein is composed of 223 amino acids with a molecular mass of 25,376 daltons, and apparently is stabilized by eight disulfide bridges. The peptide sequence showed significant homology with a family of cysteine-rich secretory proteins found in the male genital tract and in salivary glands. The interaction of helothermine and ryanodine receptors should serve to define functional domains within the channel structure involved in the control of Ca2+ release from sarcoplasmic reticulum.

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein‐C (cMyBP‐C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca2+ sensitivity of force (pCa50), PKA treatment has been shown to decrease pCa50, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca2+‐independent force and maximum Ca2+‐activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase‐specific differential effects on steady‐state force, we used synchrotron low‐angle X‐ray diffraction to compare equatorial intensity ratios (I1,1/I1,0) to assess the proximity of myosin cross‐bridge mass relative to actin and to compare lattice spacings (d1,0) to assess the inter‐thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP‐C increases I1,1/I1,0 and, as hypothesized, treatment with MLCK also increased I1,1/I1,0, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by ∼2 nm (Δ 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP‐C phosphorylation increases the proximity of cross‐bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP‐C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca2+ sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

Role of myosin heavy chain composition in kinetics of force development and relaxation in rat myocardium

1 The effects of ventricular myosin heavy chain (MHC) composition on the kinetics of activation and relaxation were examined in both chemically skinned and intact myocardial preparations from adult rats. Thyroid deficiency was induced to alter ventricular MHC isoform expression from ∼80 %α‐MHC/20 %β‐MHC in euthyroid rats to 100 %β‐MHC, without altering the expression of thin‐filament‐associated regulatory proteins. 2 In single skinned myocytes, increased expression of β‐MHC did not significantly affect either maximal Ca2+‐activated tension (P0) or the Ca2+ sensitivity of tension (pCa50). However, unloaded shortening velocity (V0) decreased by 80 % due to increased β‐MHC expression. 3 The kinetics of activation and relaxation were examined in skinned multicellular preparations using the caged Ca2+ compound DM‐nitrophen and caged Ca2+ chelator diazo‐2, respectively. Myocardium expressing 100 %β‐MHC exhibited apparent rates of submaximal and maximal tension development (kCa) that were 60 % lower than in control myocardium, and a 2‐fold increase in the half‐time for relaxation from steady‐state submaximal force. 4 The time courses of cell shortening and intracellular Ca2+ transients were assessed in living, electrically paced myocytes, both with and without β‐adrenergic stimulation (70 nm isoproterenol (isoprenaline)). Thyroid deficiency had no affect on either the extent of myocyte shortening or the resting or peak fura‐2 fluorescence ratios. However, induction of β‐MHC expression by thyroid deficiency was associated with increased half‐times for myocyte shortening and relengthening and increased half‐time for the decay of the fura‐2 fluorescence ratio. Qualitatively similar results were obtained in both the absence and the presence of β‐adrenergic stimulation although the β‐agonist accelerated the kinetics of the twitch and the Ca2+ transient. 5 Collectively, these data provide evidence that increased β‐MHC expression contributes significantly to the observed depression of contractile function in thyroid deficient myocardium by slowing the rates of both force development and force relaxation.

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.

Cross-bridge interaction kinetics in rat myocardium are accelerated by strong binding of myosin to the thin filament

1 To determine the ability of strong‐binding myosin cross‐bridges to activate the myocardial thin filament, we examined the Ca2+ dependence of force and cross‐bridge interaction kinetics at 15°C in the absence and presence of a strong‐binding, non‐force‐generating derivative of myosin subfragment‐1 (NEM‐S1) in chemically skinned myocardium from adult rats. 2 Relative to control conditions, application of 6 μM NEM‐S1 significantly increased Ca2+‐independent tension, measured at pCa 9.0, from 0.8 ± 0.3 to 3.7 ± 0.8 mN mm−2. Furthermore, NEM‐S1 potentiated submaximal Ca2+‐activated forces and thereby increased the Ca2+ sensitivity of force, i.e. the [Ca2+] required for half‐maximal activation (pCa50) increased from pCa 5.85 ± 0.05 to 5.95 ± 0.04 (change in pCa50 (ΔpCa50) = 0.11 ± 0.02). The augmentation of submaximal force by NEM‐S1 was accompanied by a marked reduction in the steepness of the force‐pCa relationship for forces less than 0.50 Po (maximum Ca2+‐activated force), i.e. the Hill coefficient (n2) decreased from 4.72 ± 0.38 to 1.54 ± 0.07. 3 In the absence of NEM‐S1, the rate of force redevelopment (ktr) was found to increase from 1.11 ± 0.21 s−1 at submaximal [Ca2+] (pCa 6.0) to 9.28 ± 0.41 s−1 during maximal Ca2+ activation (pCa 4.5). Addition of NEM‐S1 reduced the Ca2+ dependence of ktr by eliciting maximal values at low levels of Ca2+, i.e. ktr was 9.38 ± 0.30 s−1 at pCa 6.6 compared to 9.23 ± 0.27 s−1 at pCa 4.5. At intermediate levels of Ca2+, ktr was less than maximal but was still greater than values obtained at the same pCa in the absence of NEM‐S1. 4 NEM‐S1 dramatically reduced both the extent and rate of relaxation from steady‐state submaximal force following flash photolysis of the caged Ca2+ chelator diazo‐2. 5 These data demonstrate that strongly bound myosin cross‐bridges increase the level of thin filament activation in myocardium, which is manifested by an increase in the rate of cross‐bridge attachment, potentiation of force at low levels of free Ca2+, and slowed rates of relaxation.

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.

Aging-dependent depression in the kinetics of force development in rat skinned myocardium

Normal aging of the rodent heart results in prominent prolongation of the twitch. We tested the hypothesis that increased expression of beta-myosin heavy chain (MHC), as occurs in the normal aging process in the rodent heart, contributes to the prolongation of the twitch by depressing the kinetics of cross-bridge interaction. Using 3-, 9-, 21-, and 33-mo-old male Fischer 344 x Brown Norway F1 hybrid rats, we examined both the rate of tension development (kCa) and unloaded shortening velocity in chemically skinned myocardium. Although kCa in all four age groups was dependent on the level of Ca2+ activation, both submaximal and maximal kCa were significantly slower in 9-, 21-, and 33-mo-old rats relative to 3-mo-old rats. Furthermore, unloaded shortening velocity was significantly reduced in 9-, 21-, and 33-mo-old rats compared with 3-mo-old rats. Collectively, these data strongly suggest that the aging-related increase in beta-MHC expression results in a progressive slowing of cross-bridge interaction kinetics in skinned myocardium, which most likely contributes to the overall aging-dependent reduction in myocardial functional capacity.Normal aging of the rodent heart results in prominent prolongation of the twitch. We tested the hypothesis that increased expression of β-myosin heavy chain (MHC), as occurs in the normal aging process in the rodent heart, contributes to the prolongation of the twitch by depressing the kinetics of cross-bridge interaction. Using 3-, 9-, 21-, and 33-mo-old male Fischer 344 × Brown Norway F1hybrid rats, we examined both the rate of tension development ( k Ca) and unloaded shortening velocity in chemically skinned myocardium. Although k Ca in all four age groups was dependent on the level of Ca2+ activation, both submaximal and maximal k Cawere significantly slower in 9-, 21-, and 33-mo-old rats relative to 3-mo-old rats. Furthermore, unloaded shortening velocity was significantly reduced in 9-, 21-, and 33-mo-old rats compared with 3-mo-old rats. Collectively, these data strongly suggest that the aging-related increase in β-MHC expression results in a progressive slowing of cross-bridge interaction kinetics in skinned myocardium, which most likely contributes to the overall aging-dependent reduction in myocardial functional capacity.

Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers

To determine the role of myosin regulatory light chain (RLC) in modulating contraction in skeletal muscle, we examined the rate of tension development in bundles of skinned skeletal muscle fibers as a function of the level of Ca(2+) activation after UV flash-induced release of Ca(2+) from the photosensitive Ca(2+) chelator DM-nitrophen. In control fiber bundles, the rate of tension development was highly dependent on the concentration of activator Ca(2+) after the flash. There was a greater than twofold increase in the rate of tension development when the post-flash [Ca(2+)] was increased from the lowest level tested (which produced a steady tension that was 42% of maximum tension) to the highest level (producing 97% of maximum tension). However, when 40-70% of endogenous myosin RLC was extracted from the fiber bundles, tension developed at the maximum rate, regardless of the post-flash concentration of Ca(2+). Thus, the Ca(2+) dependence of the rate of tension development was eliminated by partial extraction of myosin RLC, an effect that was partially reversed by recombination of RLC back into the fiber bundles. The elimination of the Ca(2+) dependence of the kinetics of tension development was specific to the extraction of RLC rather than an artifact of the co-extraction of both RLC and Troponin C, because the rate of tension development was still Ca(2+) dependent, even when nearly 50% of endogenous Troponin C was extracted from fiber bundles fully replete with RLC. Thus, myosin RLC appears to be a key component in modulating Ca(2+) sensitive cross-bridge transitions that limit the rate of force development after photorelease of Ca(2+) in skeletal muscle fibers.