Decreasing Tropomyosin Phosphorylation Rescues Tropomyosin-induced Familial Hypertrophic Cardiomyopathy

Schulz, Emily M.; Wilder, Tanganyika; Chowdhury, Shamim; Sheikh, Hajer N.; Wolska, Beata M.; Solaro, R. John; Wieczorek, David F.

doi:10.1074/jbc.m113.466466

Cited by 23 publications

(24 citation statements)

References 57 publications

(77 reference statements)

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“…Nevertheless, considering drastic physiological alterations occur with phosphorylation of other cardiac contractile proteins, it is somewhat surprising that phosphorylation of Tpm appears to exhibit a more modest effect on cardiac function. Because previous studies determined that Tpm phosphorylation is altered during both hypertrophic and dilated cardiomyopathic conditions (16,31), studies are currently in progress to determine whether this post-translational process plays a role in ameliorating or augmenting the response to cardiomyopathic diseases.…”

Section: Discussionmentioning

confidence: 99%

“…Additional studies examined whether decreasing Tpm phosphorylation may improve cardiac function in the context of a chronic, intrinsic stressor. To examine this hypothesis, we generated transgenic mice that incorporated both the S283A mutation and a cardiac hypertrophic E180G mutation (18,31); the result- A and B) or immunoprecipitated with CK2 (C and D). Note Tpm was not stripped off membrane in A prior to probing for CK2 in B; thus, both bands are present in B. E, yeast twohybrid assay of Tpm with CK2.…”

Section: Discussionmentioning

confidence: 99%

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Tropomyosin pseudo-phosphorylation results in dilated cardiomyopathy

Rajan

Jagatheesan

Petrashevskaya

et al. 2019

Journal of Biological Chemistry

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Edited by Velia M. FowlerPhosphorylation of cardiac sarcomeric proteins plays a major role in the regulation of the physiological performance of the heart. Phosphorylation of thin filament proteins, such as troponin I and T, dramatically affects calcium sensitivity of the myofiber and systolic and diastolic functions. Phosphorylation of the regulatory protein tropomyosin (Tpm) results in altered biochemical properties of contraction; however, little is known about the physiological effect of Tpm phosphorylation on cardiac function. To address the in vivo significance of Tpm phosphorylation, here we generated transgenic mouse lines having a phosphomimetic substitution in the phosphorylation site of ␣-Tpm (S283D). High expression of Tpm S283D variant in one transgenic mouse line resulted in an increased heart:body weight ratio, coupled with a severe dilated cardiomyopathic phenotype resulting in death within 1 month of birth. Moderate Tpm S283D mice expression in other lines caused mild myocyte hypertrophy and fibrosis, did not affect lifespan, and was coupled with decreased expression of extracellular signal-regulated kinase 1/2 kinase signaling. Physiological analysis revealed that the transgenic mice exhibit impaired diastolic function, without changes in systolic performance. Surprisingly, we observed no alterations in calcium sensitivity of the myofibers, cooperativity, or calcium-ATPase activity in the myofibers. Our experiments also disclosed that casein kinase 2 plays an integral role in Tpm phosphorylation. In summary, increased expression of pseudophosphorylated Tpm impairs diastolic function in the intact heart, without altering calcium sensitivity or cooperativity of myofibers. Our findings provide the first extensive in vivo assessment of Tpm phosphorylation in the heart and its functional role in cardiac performance.Phosphorylation plays a major role in the regulation of cardiac function by affecting numerous membrane, cytoplasmic, and sarcomeric proteins. In the heart, phosphorylation of cardiac troponin I, cardiac troponin T, myosin-binding protein C, and titin regulate myofibrillar contraction, relaxation, and cross-bridge cycling (1-3). In contrast, relatively little is known regarding the importance of tropomyosin (Tpm) 8 phosphorylation. Previous investigations established that Tpm is phosphorylated at serine 283, the penultimate amino acid of the protein (4 -8). Tpm phosphorylation in the heart is developmentally regulated, with phosphorylated ␣-Tpm being the predominant isoform during fetal and newborn stages (ϳ60 -70%); in the adult mouse heart, the level of ␣-Tpm phosphorylation is decreased to ϳ30% (3). To address this developmental decrease in cardiac Tpm phosphorylation, we previously found there is a continuous decrease after birth until 5 months, whereupon increases occur by 15 months (5). We, and others, determined there is differential Tpm phosphorylation within the four cardiac chambers, with atria having the highest levels of phosphorylated Tpm (9). 9 In vitro investigations by Heel...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Tropomyosin pseudo-phosphorylation results in dilated cardiomyopathy

Rajan

Jagatheesan

Petrashevskaya

et al. 2019

Journal of Biological Chemistry

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“…While the PKA activity is usually depressed in the late stage of human heart failure, 29, 30 there are reports showing preserved or elevated PKA phosphorylation on TnI and PLB. 31, 32 It is not completely understood how the cellular PKA activity in myocytes evolves during the course of heart failure development.…”

Section: Discussionmentioning

confidence: 99%

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure

Barbagallo

Reddy

et al. 2016

Circulation Research

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Rationale In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear. Objective This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure. Methods and Results We generate a novel genetically encoded protein kinase A (PKA) biosensor anchored onto the myofilaments in rabbit cardiac myocytes to examine PKA activity at the myofilaments in responses to adrenergic stimulation. We show that PKA activity is shifted from the sarcolemma to the myofilaments in hypertrophic failing rabbit myocytes. In particular, the increased PKA activity on the myofilaments is due to an enhanced β2 adrenergic receptor (β2AR) signal selectively directed to the myofilaments together with a reduced phosphodiesterase activity associated with the myofibrils. Mechanistically, the enhanced PKA activity on the myofilaments is associated with downregulation of caveolin-3 in the hypertrophic failing rabbit myocytes. Reintroduction of caveolin-3 in the failing myocytes is able to normalize the distribution of β2AR signal by preventing PKA signal access to the myofilaments, and to restore contractile response to adrenergic stimulation. Conclusions In hypertrophic rabbit myocytes, selectively enhanced β2AR signaling toward the myofilaments contributes to elevated PKA activity and PKA phosphorylation of myofilament proteins. Reintroduction of caveolin-3 is able to confine β2AR signaling and restore myocyte contractility in response to β-adrenergic stimulation.

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“…references 29, 44]. Phosphorylation-induced enhanced thin filament responsiveness to narrower cellular Ca 2+ concentrations ranges will be advantageous to fetal muscle, where the control of intracellular Ca 2+ -levels may not be fully developed [45–47], and provide compensatory mechanisms in myopathic muscle [48], where Ca 2+ metabolism may be compromised.…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation of Ser283 enhances the stiffness of the tropomyosin head-to-tail overlap domain

Lehman

Medlock

et al. 2015

Archives of Biochemistry and Biophysics

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The ends of coiled-coil tropomyosin molecules are joined together by nine to ten residue-long head-to-tail “overlapping domains”. These short four-chained interconnections ensure formation of continuous tropomyosin cables that wrap around actin filaments. Molecular Dynamics simulations indicate that the curvature and bending flexibility at the overlap is 10 to 20% greater than over the rest of the molecule, which might affect head-to-tail filament assembly on F-actin. Since the penultimate residue of striated muscle tropomyosin, Ser283, is a natural target of phosphorylating enzymes, we have assessed here if phosphorylation adjusts the mechanical properties of the tropomyosin overlap domain. MD simulations show that phosphorylation straightens the overlap to match the curvature of the remainder of tropomyosin while stiffening it to equal or exceed the rigidity of canonical coiled-coil regions. Corresponding EM data on phosphomimetic tropomyosin S283D corroborate these findings. The phosphorylation-induced change in mechanical properties of tropomyosin likely results from electrostatic interactions between C-terminal phosphoSer283 and N-terminal Lys12 in the four-chain overlap bundle, while promoting stronger interactions among surrounding residues and thus facilitating tropomyosin cable assembly. The stiffening effect of D283-tropomyosin noted correlates with previously observed enhanced actin-tropomyosin activation of myosin S1-ATPase, suggesting a role for the tropomyosin phosphorylation in potentiating muscle contraction.

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Decreasing Tropomyosin Phosphorylation Rescues Tropomyosin-induced Familial Hypertrophic Cardiomyopathy

Cited by 23 publications

References 57 publications

Tropomyosin pseudo-phosphorylation results in dilated cardiomyopathy

Tropomyosin pseudo-phosphorylation results in dilated cardiomyopathy

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure

Phosphorylation of Ser283 enhances the stiffness of the tropomyosin head-to-tail overlap domain

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