Phosphorylation Of Myofibrillar Proteins Research Articles

Both PKA Treatment and Cardiac Troponin-I N-Terminal Phosphorylation Alone Decrease Ca-Sensitivity and Eliminate Length-Dependent Activation in Skinned Cardiac Muscle

Protein kinase A (PKA) phosphorylation of myofibrillar proteins constitutes an important pathway for β-adrenergic modulation of cardiac contractility. PKA targets the cardiac troponin I (cTnI) N-terminus, cardiac myosin-binding protein C (cMyBP-C) and titin. To isolate cTnI and cMyBP-C /titin phosphorylation effects on force-[Ca2+] relations, endogenous cardiac troponin (Tn) was exchanged in rat, skinned trabeculae with either WT Tn or Tn containing a non-phosphorylatable mutant cTnI(S23/24A) or phosphomimetic cTnI(S23/24D). PKA cannot phosphorylate either cTnI mutant, leaving cMyBP-C and titin as sole PKA targets. Force-[Ca2+] relations and Ca2+-sensitivity (pCa50) were measured at 2.3 and 2.0 µm SL. Decreasing steady SL reduced maximal force (Fmax) and pCa50 similarly with WT Tn and Tn containing cTnI(S23/24A). PKA treatment of native, WT and cTnI(S23/24A) exchanged trabeculae reduced pCa50 at 2.3, but not 2.0 um SL, eliminating SL-dependence of pCa50. Reconstitution with Tn containing cTnI(S23/24D) reduced pCa50 at both SL (compared to WT and cTnI(S23,24A) and eliminated pCa50 SL-dependence; PKA did not significantly alter pCa50 at either SL. At each SL Fmax was similar with WT and mutant troponins, and was unaffected by PKA. Low angle x-ray diffraction experiments were performed to determine whether shifts in pCa50 were associated with changes in myofilament spacing (D1,0) or interaction. D1,0 at 2.3 um SL was similar in native trabeculae, with WT Tn and Tn containing either cTnI(S23,24A) or cTnI(S23,24D); PKA increased D1,0 in all cases. The results suggest that PKA phosphorylation of either cTnI or cMyBP-C /titin reduced the Ca2+-sensitivity of force and length-dependent activation. Supported by NIH HL067071-06.

Titin Phosphorylation

See related articles, pages 631–638 Diastolic performance is regulated by net myocardial stiffness, which is determined by the mechanosensitive protein network comprised of extracellular proteins such as collagen, intracellular sarcomeric proteins, and cell surface integrins. Mechanical force and its regulation are sensed and propagated by each of these components in a concerted fashion.1 During diastole, titin filaments serve as tensiometers and passive force generators. This is accomplished by modulation via directional signaling of multiple linkages between different regions within the titin molecule and the cellular contractile apparatus. An intricate network of signaling molecules coordinates the extracellular and intracellular components in the contraction of a sarcomere. In this issue of Circulation Research , Hidalgo et al present an elegant set of experiments that reveal a novel mechanism whereby altering the phosphorylation state of titin modulates myocardial passive stiffness.2 Specifically, they demonstrated that titin, in addition to being a protein kinase (PK)A and PKG substrate, is also a PKCα substrate. They further identified the PEVK region of titin as the prominent site of PKCα phosphorylation, and showed that phosphorylation at this site increased passive tension. Titin is an approximately 3000-kDa molecular-mass sarcomeric protein that spans the Z disk to the M line of the sarcomere.3 Originally thought to only provide structural scaffolding to link the many regulatory, contractile, and structural proteins within the sarcomere, titin is now recognized to be a major regulator of intracellular myocyte stiffness. Titin determines the passive tension of the intracellular component of cardiomyocytes, which, together with collagen, determines the total myocardial passive stiffness. Although the immunoglobulin-like domain and fibronectin type III repeats make up 90% of the titin molecule, titin also includes a unique I-band region that is flexible and specifically serves as a molecular spring to determine passive stiffness. …

Sarcomere length dependence of power output is increased after PKA treatment in rat cardiac myocytes

The Frank-Starling relationship of the heart yields increased stroke volume with greater end-diastolic volume, and this relationship is steeper after beta-adrenergic stimulation. The underlying basis for the Frank-Starling mechanism involves length-dependent changes in both Ca(2+) 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 beta-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 Ca(2+) sensitivity of force and increased loaded shortening and power output at all relative loads when the myocyte preparations were at long sarcomere length ( approximately 2.30 mum). PKA had less of an effect on loaded shortening and power output at short sarcomere length ( approximately 2.0 mum). These changes resulted in a greater length dependence of myocyte power output after PKA treatment; peak normalized power output increased approximately 20% with length before PKA and approximately 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 beta-adrenergic stimulation of the left ventricle.

Abstract 234: Expression of Activated PKCϵ Accelerates the Transition to Heart Failure in Response to Increased Hemodynamic Load

Expression of constitutively active PKCϵ in the myocardium promotes a steady progression to a dilated cardiomyopathy over time. Functional and biochemical studies demonstrate that the myofibrillar proteins are a target of PKCϵ mediated phosphorylation. In this model, changes in myofibrillar protein phosphorylation at 3-months are associated with compensatory mechanisms, whereas at 9 and 12-months are associated with the transition to failure. To test the hypothesis that PKCϵ phosphorylation alters the post-translational modification of the contractile proteins and that these changes predispose the heart to failure, we imposed a hemodynamic load on young mice (3-mice). Both transgenic (TG) and non-transgenic (NTG) mice were banded (AoB) for 10 weeks before assessment of cardiac function in vivo , gene expression and 2-D electrophoresis of myofibrillar extracts. Analysis of heart weight to body weight ratios (3mNTG=4.3±0.4 vs. 3mTG=4.2±0.3 vs. 3mNTG(AoB)= 5.9±0.3* vs. 3mT-G(AoB)=7.4±0.6** *p<0.05), shows hypertrophy in both groups of AoB mice, but to a greater degree in the TG (AoB). Gene expression analysis using quantitative qRT-PCR shows significant increases in both ANF and β-MHC mRNA in AoB mice compared with non-banded groups. Expression was significantly greater in the TG (AoB) along with a decrease in SERCA mRNA. Banding induced both diastolic and systolic dysfunction in TG compared to NTG hearts. Maximal −dP/dt in the TG (AoB) hearts was significantly lower compared to all other groups (3mNTG=9433±1000 vs. 3mTG=9474±857 vs. 3mNTG(AoB)=8620±1264 vs. 3mT-G(AoB)=4911±738* mmHg/sec, *p<0.05). Load-independent measurements revealed a significant decreases the time constant of isovolumic relaxation (3mNTG(AoB)=5.4±0.6 vs. 3mTG(AoB)=8.4±1.5* ms). In addition, maximal +dP/dt-end diastolic volume relation (3mNTG(AoB)=213±64 vs. 3mTG(AoB)=79±20*, p<0.05) was significantly depressed in banded TG compared to banded NTG. 2-D gel analysis of myofibrillar proteins revealed hyper-phosphorylation of troponin I in the PKCϵ TG mice. These data suggest that while the TG hearts have compensated to an initial insult (PKC activation) imposition of a second insult (hemodynamic load) accelerates the transition to failure.

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 (cTnI(ala2)), or both (cMyBP-C(-/-)/cTnI(ala2)), we investigated the roles of cTnI and cMyBP-C phosphorylation in the regulation of the stretch-activation response. PKA treatment of wild-type and cTnI(ala2) skinned ventricular myocardium accelerated stretch activation such that the response was indistinguishable from stretch activation of cMyBP-C(-/-) or cMyBP-C(-/-)/cTnI(ala2) myocardium; however, PKA had no effect on stretch activation in cMyBP-C(-/-) or cMyBP-C(-/-)/cTnI(ala2) myocardium. These results indicate that the acceleration of stretch activation in wild-type and cTnI(ala2) myocardium is caused by phosphorylation of cMyBP-C and not cTnI. We conclude that the primary effect of PKA phosphorylation of cTnI is reduced Ca(2+) 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.

PKC depresses cardiac myocyte power output and limits the phosphorylation and mechanical effects by PKA

Protein kinase C (PKC) targets and phosphorylates myofibrillar proteins in cardiac myocytes, and PKC activity appears to be up-regulated in failing human hearts (Bowling et al., 1999). Since PKC phosphorylation of myofibrillar proteins affects myocyte contractility, we sought to directly test the effects of PKC phosphorylation of myofilament proteins on myocyte power output. We compared isometric force, loaded shortening velocity, and power output in skinned rat cardiac myocytes before and after treatment with the catalytic subunit of PKC.

Power output is increased after phosphorylation of myofibrillar proteins in rat skinned cardiac myocytes.

beta-Adrenergic stimulation increases stroke volume in mammalian hearts as a result of protein kinase A (PKA)-induced phosphorylation of several myocyte proteins. This study investigated whether PKA-induced phosphorylation of myofibrillar proteins directly affects myocyte contractility. To test this possibility, we compared isometric force, loaded shortening velocity, and power output in skinned rat cardiac myocytes before and after treatment with the catalytic subunit of PKA. Consistent with previous studies, PKA increased phosphorylation levels of myosin binding protein C and troponin I, and reduced Ca(2+) sensitivity of force. PKA also significantly increased both maximal force (25.4+/-8.3 versus 31.6+/-11.3 microN [P<0.001, n=12]) and peak absolute power output (2.48+/-1.33 versus 3.38+/-1.52 microW/mg [P<0.05, n=5]) during maximal Ca(2+) activations. Furthermore, PKA elevated power output at nearly all loads even after normalizing for the increase in force. After PKA treatment, peak normalized power output increased approximately 20% during maximal Ca(2+) activations (n=5) and approximately 33% during half-maximal Ca(2+) activations (n=9). These results indicate that PKA-induced phosphorylation of myofibrillar proteins increases the power output-generating capacity of skinned cardiac myocytes, in part, by speeding the step(s) in the crossbridge cycle that limit loaded shortening rates, and these changes likely contribute to greater contractility in hearts after beta-adrenergic stimulation.

Altered phosphorylation and calcium sensitivity of cardiac myofibrillar proteins during sepsis.

Altered phosphorylation and Ca(2+) sensitivity of cardiac myofibrillar proteins during different phases of sepsis were investigated. Sepsis was induced by cecal ligation and puncture (CLP). The results show that phosphorylation of troponin I (TnI) was increased by 268% during the early phase (9 h after CLP) but decreased by 46% during the late phase (18 h after CLP) of sepsis. Phosphorylation of C protein was increased by 76% during the early phase but decreased by 41% during the late phase of sepsis. Phosphorylation of myosin light chain-2 (MLC-2) remained unaltered during the early phase but was decreased by 38% during the late phase of sepsis. Phosphorylation of TnT was unaffected during the progression of sepsis. The increases in the phosphorylation of TnI and C protein during early sepsis were associated with the decrease in the Ca(2+) sensitivity of myofilaments and the increases in myocardial changes in tension development (+dP/dt(max)) and cAMP level. The decreases in the phosphorylation of TnI and C protein during late sepsis coincided with the declines in the activities of myofibrillar ATPase, Ca(2+) sensitivity of myofilaments, myocardial +/-dP/dt(max), and cAMP content. The increases and the decreases in the phosphorylation of TnI and C protein, +/-dP/dt(max), and the tissue cAMP level were sensitive to isoproterenol stimulation and propranolol inhibition. These findings suggest that alterations in the phosphorylation of myofibrillar proteins, such as TnI, C protein, and MLC-2, and changes in the activities and the Ca(2+) sensitivity of myofibrillar ATPase may contribute to the altered cardiac function during the progression of sepsis. Furthermore, the sepsis-induced alterations in the phosphorylation and Ca(2+) sensitivity of cardiac myofibrillar proteins were mediated via a beta-adrenergic receptor pathway.

PKA accelerates rate of force development in murine skinned myocardium expressing alpha- or beta-tropomyosin.

In myocardium, protein kinase A (PKA) is known to phosphorylate troponin I (TnI) and myosin-binding protein-C (MyBP-C). Here, we used skinned myocardial preparations from nontransgenic (NTG) mouse hearts expressing 100% alpha-tropomyosin (alpha-Tm) to examine the effects of phosphorylated TnI and MyBP-C on Ca2+ sensitivity of force and the rate constant of force redevelopment (k(tr)). Experiments were also done using transgenic (TG) myocardium expressing approximately 60% beta-Tm to test the idea that the alpha-Tm isoform is required to observe the mechanical effects of PKA phosphorylation. Compared with NTG myocardium, TG myocardium exhibited greater Ca2+ sensitivity of force and developed submaximal forces at faster rates. Treatment with PKA reduced Ca2+ sensitivity of force in NTG and TG myocardium, had no effect on maximum k(tr) in either NTG or TG myocardium, and increased the rates of submaximal force development in both kinds of myocardium. These results show that PKA-mediated phosphorylation of myofibrillar proteins significantly alters the static and dynamic mechanical properties of myocardium, and these effects occur regardless of the type of Tm expressed.

Phosphorylation by protein kinases A and C of myofibrillar proteins in rabbit stunned and non-stunned myocardium.

We tested the hypothesis that altered phosphorylation of myofibrillar proteins is involved in post-ischemic myocardial stunning. Myofibrillar proteins were isolated from Langendorff perfused control rabbit hearts, hearts submitted to 15 min normothermic ischemia and hearts submitted to 15 min ischemia followed by 10 min of reperfusion (stunned hearts). The in vivo level of phosphorylation of specific contractile proteins by protein kinases A and C was indirectly detected by the amount of 32P incorporated in vitro in the presence of these protein kinases and saturating concentration of [gamma-32P]-ATP (back-phosphorylation method). In control experiments the back-phosphorylation technique was able to detect PKA- or PKC-induced protein phosphorylation in hearts treated with isoproterenol and phorbol ester, respectively. In stunned hearts, contractile function was significantly suppressed compared to the period before ischemia. We found no difference in myofibrillar protein profile (on densitometry of the Coomassie-stained gels after SDS-PAGE) and in PKA mediated 32P incorporation when comparing control, ischemic and stunned myocardium. Three different PKCs were used for phosphorylation: commercial purified rat brain PKC, partially purified rat brain PKC or rabbit partially purified cardiac PKC. Cardiac PKC mainly phosphorylated troponin I, whereas brain PKC phosphorylated both troponin T and troponin I. No significant difference in 32P incorporation mediated by either brain or cardiac PKC was found between control, ischemic and ischemic/reperfused myofibrils. These data indicate that myocardial stunning does not cause changes in PKC- or PKA-mediated Pi incorporation into myofibrillar proteins detectable by the back-phosphorylation method.

Experimental diabetes is associated with functional activation of protein kinase C epsilon and phosphorylation of troponin I in the heart, which are prevented by angiotensin II receptor blockade.

A cardiomyopathy that is characterized by an impairment in diastolic relaxation and a loss of calcium sensitivity of the isolated myofibril has been described in chronic diabetic animals and humans. To explore a possible role for protein kinase C (PKC)-mediated phosphorylation of myofibrillar proteins in this process, we characterized the subcellular distribution of the major PKC isoforms seen in the adult heart in cardiocytes isolated from diabetic rats and determined patterns of phosphorylation of the major regulatory proteins, including troponin I (TnI). Rats were made diabetic with a single injection of streptozotocin, and myocardiocytes were isolated and studied 3 to 4 weeks later. In nondiabetic animals, 76% of the PKC epsilon isoform was located in the cytosol and 24% was particulate, whereas in diabetic animals, 55% was cytosolic and 45% was particulate (P < .05). PKC delta, the other major PKC isoform seen in adult cardiocytes, did not show a change in subcellular localization. In parallel, TnI phosphorylation was increased 5-fold in cardiocytes isolated from the hearts of diabetic animals relative to control animals (P < .01). The change in PKC epsilon distribution and in TnI phosphorylation in diabetic animals was completely prevented by rendering the animals euglycemic with insulin or by concomitant treatment with a specific angiotensin II type-1 receptor (AT1) antagonist. Since PKC phosphorylation of TnI has been associated with a loss of calcium sensitivity of intact myofibrils, these data suggest that angiotensin II receptor-mediated activation of PKC may play a role in the contractile dysfunction seen in chronic diabetes.

Effect of preperfusion of ascending concentrations of lead on digoxin-induced cardiac arrest in isolated frog heart

The present work investigated the effect of preperfusion of ascending concentrations of lead acetate (LA) (10 −9, 10 −7 and 10 −5 M) on digoxin (DGN) cardiotoxicity in isolated frog heart, in order to look for any consequent variations in its lead-induced potentiation. The DGN perfusion time (s) and DGN exposure (μg DGN 10 mg heart weight) for, and myocardial DGN level (ng DGN g wet tissue) at, cardiac arrest were the parameters evaluated so as to assess cardiotoxicity. Both sodium acetate and LA (10 −7 M) preperfusion led to a diminution in cardiac rate at 10 min of DGN perfusion without altering the contractility compared to the DGN alone group. With regard to DGN perfusion time for cardiac arrest, preperfusion of ascending concentrations of LA induced a corresponding decrease which was statistically significant ( P <0.05). On the other hand, in the experimental group that received preperfusion of 10 −9 M LA, the DGN exposure for cardiac arrest was not significantly different from that of the control, whereas in the 10 −7 and 10 −5 M groups, it was significantly lower ( P <0.05). In the experimental group that received preperfusion of 10 −7 M LA, the significant reduction in DGN perfusion time and DGN exposure was well corroborated by a diminution in the myocardial DGN level (4.01 ± 0.17 ng g wet tissue in comparison with the control value of 5.72 ± 0.4 ng g wet tissue, P < 0.05) at cardiac arrest. Taken together, these data reveal that with the preperfusion of LA in ascending concentrations, there is a relative increase in LA-induced potentiation of DGN cardiotoxicity. The present findings may be partially explained by the reported competition between Pb 2+ and Ca 2+ ions at the cellular level, with the consequent antagonism of calcium-stimulated cardiac contractility and myofibrillar protein phosphorylation, and also by the inhibitory effect of lead on (Na +,K +)-ATPase, and binding of digitalis to this enzyme.

Inotropic responses to isoproterenol and phosphodiesterase inhibitors in intact guinea pig hearts: comparison of cyclic AMP levels and phosphorylation of sarcoplasmic reticulum and myofibrillar proteins.

The influence of selective (milrinone: 10, 50, 100 microM) and nonselective phosphodiesterase (isobutylmethylxanthine: 0.1, 10, 100 microM) inhibitors and beta-adrenergic stimulation (isoproterenol: 0.01, 0.1 microM) on phospholamban and myofibrillar protein phosphorylation was studied in guinea pig hearts perfused with [32P]orthophosphate. Changes in protein phosphorylation were compared to alterations in tissue cyclic AMP (cAMP) levels and positive inotropic effects induced by these agents. Isoproterenol (0.01 microM), milrinone (50 microM), and isobutylmethylxanthine (100 microM) all produced similar, twofold increases in dP/dt and -dP/dt but only stimulation with isobutylmethylxanthine and isoproterenol was associated with significant increases in phospholamban phosphorylation. At these equipotent doses, the effects of isobutylmethylxanthine were associated with higher increases (3.1-fold) in cAMP than those observed with isoproterenol (twofold). Milrinone (50 microM) produced a 2.5-fold increase in cAMP levels but failed to change phospholamban phosphorylation. Higher doses of milrinone (100 microM) resulted in relatively high (4.1-fold) cAMP levels, and this was associated with increased (1.5-fold) phosphorylation of phospholamban. Phosphorylation of troponin I was significantly increased at 0.01 microM and 0.1 microM isoproterenol, while phosphorylation of C protein was observed only at 0.1 microM isoproterenol. Isobutylmethylxanthine and milrinone did not significantly increase phosphorylation of either troponin I or C protein at any of the doses studied. These findings indicate that cardiotonic agents acting via the cAMP pathway may produce similar inotropic responses at different levels of cAMP and phosphorylation of sarcoplasmic reticulum and myofibrillar proteins.

Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase.

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[gamma-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143,000, 60,000, 42,000, 33,000, and 11,000. The 143,000 MW substrate was identified as C-protein; the 42,000 MW substrate is probably actin; the 33,000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60,000 and 11,000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein as phosphorylated to the extent of approximately 0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5-6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of approximately 0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin).(ABSTRACT TRUNCATED AT 250 WORDS)

Dephosphorylation of myofibrillar proteins in actomyosin preparations and in isolated perfused rat hearts after β-agonist withdrawal

The in vitro dephosphorylation of myosin P light chains and troponin-I was studied in preparations of natural actomyosin isolated after isoproterenol treatment of perfused rat hearts. These preparations contained endogenous phosphatase(s) which on incubation removed incorporated 22P from myosin P light chains more rapidly than from troponin-I. The in situ effects of isoproterenol pulse-treatment were also studied in perfused rat hearts, with respect to myofibrillar protein phosphorylation and the Ca 2+ Mg 2+ -ATPase and Ca 2+-ATPase activities of natural actomyosin. Abrupt removal of the β-adrenergic stimulus from perfused rat hearts caused the (incomplete) dephosphorylation of both myosin P light chains and troponin-I; the former was dephosphorylated in apparent synchrony with the slowing of the beating rates and falling cyclic AMP contents (definitely decreased after 1 min), whereas troponin-I dephosphorylation only began after 2 min and was correlated with changes in force development. The dephosphorylation of P light chains was accompanied by a decrease in the V max |ATP| of the myosin Ca 2+-ATPase activities, but the latter reached pre-stimulation values before basal phosphorylation levels of the protein were attained. An increase in the V max |Ca 2+| of Ca 2+ Mg 2+ -ATPase activities and an increase in their sensitivity to Ca 2+ was correlatable with the removal of phosphate from troponin-I. The results suggest that the phosphorylations of troponin-I and myosin light chains in adrenergically stimulated hearts serve separate functions in the context of positive inotropism.

Phosphorylation and adenosine triphosphatase activity of myofibrils from thyrotoxic rabbit hearts.

Cardiac hypertrophy induced by thyrotoxic stress leads to an increase in the rate of force development, velocity of shortening, tension-dependent heat generation, and myosin ATPase activity. We did studies to see whether alterations in covalent phosphorylation of myofibrillar proteins correlate with these changes. The protein preparations were isolated from control and thyrotoxic hearts of male albino rabbits freeze-clamped in situ. We measured myofibrillar ATPase, and the covalent phosphate content of ventricular myosin 19,000 (mol wt) light chain (P-light chain) and troponin I (TnI). The myofibrillar ATPase activity was increased 2-fold in the thyrotoxic preparations with no change in the level of myofibrillar phosphorylation. The covalent phosphate content of TnI was 1.21 +/- 0.09 mol P/mol TnI in control hearts and 1.14 +/- 0.04 mol P/mol TnI in thyrotoxic hearts. The covalent phosphate content of the light chain fraction was 0.41 +/- 0.06 mol P/mol P-light chain in control hearts and 0.37 +/- 0.04 mol P/mol P-light chain in thyrotoxic hearts. The dependence of the normalized myofibrillar ATPase on free calcium concentration was the same in control and thyrotoxic preparations. Thus the mechanical, thermal, and biochemical changes found in hearts from thyrotoxic animals probably occur with no change in phosphorylation of TnI or myosin light chains.

Influence of isoproterenol and calcium on cadmium- or lead-induced negative inotropy related to cardiac myofibrillar protein phosphorylations in perfused rat heart

The individual and combined physiological and myofibrillar protein phosphorylation effects of the negative inotropic agents, cadmium (3 × 10 −3 m m) and lead (3 × 10 −4 m m), and the positive inotropic agents isoproterenol (7 × 10 −7 m) and calcium (6.5 m m) were examined in isolated modified perfused rat heart preparations to evaluate possible causal associations between cadmium- and lead-induced changes in myofibrillar protein phosphorylations and altered inotropic responsiveness. Cadmium and lead alone depressed cardiac contractility and only the phosphorylation of the myosin light chain-2 (LC-2). These effects were attenuated in the presence of isoproterenol and elevated extracellular calcium; however, cadmium and lead inhibited both the positive inotropic activation of the heart by calcium and isoproterenol and the concomitant increase in phosphorylations of the purported cardioregulatory phospho-proteins, LC-2 and the troponin inhibitory subunit (TN-I). Positive chronotropic responses to the β-adrenergic agonist were unaffected by cadmium and lead. These results suggest that the negative inotropic effects of cadmium and lead are related to depressed phosphorylation of LC-2 and TN-I. Furthermore, the present findings suggest that cadmium and lead may alter myofibrillar protein phosphorylation mechanisms by antagonizing sarcolemmal calcium translocation processes; however, these results do not preclude the possibility of direct intracellular antagonisms of calcium-dependent processes by these heavy metal ions.