Increased cross-bridge cycling kinetics after exchange of C-terminal truncated troponin I in skinned rat cardiac muscle

The precise mechanism of cardiac troponin I (cTnI) proteolysis in myocardial stunning is not fully understood. Accordingly, we determined the effect of cTnI C terminus truncation on chemo-mechanical transduction in isolated skinned rat trabeculae. Recombinant troponin complex (cTn), containing either mouse cTnI-(1-193) or human cTnI-(1-192) was exchanged into skinned cardiac trabeculae; Western blot analysis confirmed that 60-70% of the endogenous cTn was replaced by recombinant Tn. Incorporation of truncated cTnI induced significant reductions ( approximately 50%) in maximum force and cooperative activation as well as increases ( approximately 50%) in myofilament Ca(2+) sensitivity and tension cost. Similar results were obtained with either mouse or human truncated cTn. Presence of truncated cTnI increased maximum actin-activated S1 ATPase activity as well as its Ca(2+) sensitivity in vitro. Partial exchange (50%) for truncated cTnI resulted in similar reductions in maximum force and cooperativity; tension cost was increased in proportion to truncated cTnI content. In vitro, to determine the molecular mechanism responsible for the enhanced myofilament Ca(2+) sensitivity, we measured Ca(2+) binding to cTn as reported using a fluorescent probe. Incorporation of truncated cTnI did not affect Ca(2+) binding affinity to cTn alone. However, when cTn was incorporated into thin filaments, cTnI truncation induced a significant increase in Ca(2+) binding affinity to cTn. We conclude that cTnI truncation induces depressed myofilament function. Decreased cardiac function after ischemia/reperfusion injury may directly result, in part, from proteolytic degradation of cTnI, resulting in alterations in cross-bridge cycling kinetics.     

Effects of potassium on vanadate inhibition of sarcoplasmic reticulum Ca2+-ATPase from dog cardiac and rabbit skeletal muscle.

The concentration of vanadate for half maximal inhibition of dog cardiac and rabbit skeletal SR Ca²⁺-ATPase was approximately 5 μM. Preincubation of the enzyme with vanadate resulted in greater inhibition. Effects of potassium on the inhibition were studied under various conditions.     

Computational Studies of the Effect of the S23D/S24D Troponin I Mutation on Cardiac Troponin Structural Dynamics

During β-adrenergic stimulation, cardiac troponin I (cTnI) is phosphorylated by protein kinase A (PKA) at sites S23/S24, located at the N-terminus of cTnI. This phosphorylation has been shown to decrease K_Ca and pCa₅₀, and weaken the cTnC-cTnI (C-I) interaction. We recently reported that phosphorylation results in an increase in the rate of early, slow phase of relaxation (k_REL,slow) and a decrease in its duration (t_REL,slow), which speeds up the overall relaxation. However, as the N-terminus of cTnI (residues 1–40) has not been resolved in the whole cardiac troponin (cTn) structure, little is known about the molecular-level behavior within the whole cTn complex upon phosphorylation of the S23/S24 residues of cTnI that results in these changes in function. In this study, we built up the cTn complex structure (including residues cTnC 1–161, cTnI 1–172, and cTnT 236–285) with the N-terminus of cTnI. We performed molecular-dynamics (MD) simulations to elucidate the structural basis of PKA phosphorylation-induced changes in cTn structure and Ca²⁺ binding. We found that introducing two phosphomimic mutations into sites S23/S24 had no significant effect on the coordinating residues of Ca²⁺ binding site II. However, the overall fluctuation of cTn was increased and the C-I interaction was altered relative to the wild-type model. The most significant changes involved interactions with the N-terminus of cTnI. Interestingly, the phosphomimic mutations led to the formation of intrasubunit interactions between the N-terminus and the inhibitory peptide of cTnI. This may result in altered interactions with cTnC and could explain the increased rate and decreased duration of slow-phase relaxation seen in myofibrils.     

Decreasing Tropomyosin Phosphorylation Rescues Tropomyosin-induced Familial Hypertrophic Cardiomyopathy

Studies indicate that tropomyosin (Tm) phosphorylation status varies in different mouse models of cardiac disease. Investigation of basal and acute cardiac function utilizing a mouse model expressing an α-Tm protein that cannot be phosphorylated (S283A) shows a compensated hypertrophic phenotype with significant increases in SERCA2a expression and phosphorylation of phospholamban Ser-16 (Schulz, E. M., Correll, R. N., Sheikh, H. N., Lofrano-Alves, M. S., Engel, P. L., Newman, G., Schultz Jel, J., Molkentin, J. D., Wolska, B. M., Solaro, R. J., and Wieczorek, D. F. (2012) J. Biol. Chem. 287, 44478–44489). With these results, we hypothesized that decreasing α-Tm phosphorylation may be beneficial in the context of a chronic, intrinsic stressor. To test this hypothesis, we utilized the familial hypertrophic cardiomyopathy (FHC) α-Tm E180G model (Prabhakar, R., Boivin, G. P., Grupp, I. L., Hoit, B., Arteaga, G., Solaro, R. J., and Wieczorek, D. F. (2001) J. Mol. Cell. Cardiol. 33, 1815–1828). These FHC hearts are characterized by increased heart:body weight ratios, fibrosis, increased myofilament Ca²⁺ sensitivity, and contractile defects. The FHC mice die by 6–8 months of age. We generated mice expressing both the E180G and S283A mutations and found that the hypertrophic phenotype was rescued in the α-Tm E180G/S283A double mutant transgenic animals; these mice exhibited no signs of cardiac hypertrophy and displayed improved cardiac function. These double mutant transgenic hearts showed increased phosphorylation of phospholamban Ser-16 and Thr-17 compared with the α-Tm E180G mice. This is the first study to demonstrate that decreasing phosphorylation of tropomyosin can rescue a hypertrophic cardiomyopathic phenotype.     

Deletion of P21-activated kinase-1 induces age-dependent increased visceral adiposity and cardiac dysfunction in female mice

It is known that there is an age-related progression in diastolic dysfunction, especially prevalent in postmenopausal women, who develop heart failure with preserved ejection fraction (HFpEF, EF?>?50%). Mechanisms and therapies are poorly understood, but there are strong correlations between obesity and HFpEF. We have tested the hypothesis that P21-activated kinase-1 (PAK1) preserves cardiac function and adipose tissue homeostasis during aging in female mice. Previous demonstrations in male mice by our lab that PAK1 activity confers cardio-protection against different stresses formed the rationale for this hypothesis. Our studies compared young (3–6 months) and middle-aged (12–15 months) female and male PAK1 knock-out mice (PAK1^?/?) and wild-type (WT) equivalent. Female WT mice exhibited increased cardiac PAK1 abundance during aging. By echocardiography, compared to young WT female mice, middle-aged WT female mice showed enlargement of the left atrium as well as thickening of posterior wall and increased left ventricular mass; however, all contraction and relaxation parameters were preserved during aging. Compared to WT controls, middle-aged PAK1^?/? female mice demonstrated worsening of cardiac function involving a greater enlargement of the left atrium, ventricular hypertrophy, and diastolic dysfunction. Moreover, with aging PAK1^?/? female mice, unlike male PAK1^?/? mice, exhibited increased adiposity with increased accumulation of visceral adipose tissue. Our data provide evidence for the significance of PAK1 signaling as an element in the preservation of cardiac function and adipose tissue homeostasis in females during aging.

     

Truncation of the N-terminus of cardiac troponin I initiates adaptive remodeling of the myocardial proteosome via phosphorylation of mechano-sensitive signaling pathways

Molecular and Cellular Biochemistry - The cardiac isoform of troponin I has a unique N-terminal extension (~?1–30 amino acids), which contributes to the modulation of cardiac...     

Tropomyosin Dephosphorylation Results in Compensated Cardiac Hypertrophy

Phosphorylation of tropomyosin (Tm) has been shown to vary in mouse models of cardiac hypertrophy. Little is known about the in vivo role of Tm phosphorylation. This study examines the consequences of Tm dephosphorylation in the murine heart. Transgenic (TG) mice were generated with cardiac specific expression of α-Tm with serine 283, the phosphorylation site of Tm, mutated to alanine. Echocardiographic analysis and cardiomyocyte cross-sectional area measurements show that α-Tm S283A TG mice exhibit a hypertrophic phenotype at basal levels. Interestingly, there are no alterations in cardiac function, myofilament calcium (Ca²⁺) sensitivity, cooperativity, or response to β-adrenergic stimulus. Studies of Ca²⁺ handling proteins show significant increases in sarcoplasmic reticulum ATPase (SERCA2a) protein expression and an increase in phospholamban phosphorylation at serine 16, similar to hearts under exercise training. Compared with controls, the decrease in phosphorylation of α-Tm results in greater functional defects in TG animals stressed by transaortic constriction to induce pressure overload-hypertrophy. This is the first study to investigate the in vivo role of Tm dephosphorylation under both normal and cardiac stress conditions, documenting a role for Tm dephosphorylation in the maintenance of a compensated or physiological phenotype. Collectively, these results suggest that modification of the Tm phosphorylation status in the heart, depending upon the cardiac state/condition, may modulate the development of cardiac hypertrophy.     

Tropomyosin 3 expression leads to hypercontractility and attenuates myofilament length-dependent Ca(2+) activation

Tropomyosin (TM), an integral component of the thin filament, is encoded by three striated muscle isoforms: alpha-TM, beta-TM, and TPM 3. Although the alpha-TM and beta-TM isoforms are well characterized, less is known about the function of the TPM 3 isoform, which is predominantly found in the slow-twitch musculature of mammals. To determine its functional significance, we ectopically expressed this isoform in the hearts of transgenic mice. We generated six transgenic mouse lines that produce varying levels of TPM 3 message with ectopic TPM 3 protein accounting for 40-60% of the total striated muscle tropomyosin. The transgenic mice have normal life spans and exhibit no morphological abnormalities in their sarcomeres or hearts. However, there are significant functional alterations in cardiac performance. Physiological assessment of these mice by using closed-chest analyses and a work-performing model reveals a hyperdynamic effect on systolic and diastolic function. Analysis of detergent-extracted fiber bundles demonstrates a decreased sensitivity to Ca(2+) in force generation and a decrease in length-dependent Ca(2+) activation with no detectable change in interfilament spacing as determined by using X-ray diffraction. Our data are the first to demonstrate that TM isoforms can affect sarcomeric performance by decreasing sensitivity to Ca(2+) and influencing the length-dependent Ca(2+) activation.     

Ca(2+) activation and tension cost in myofilaments from mouse hearts ectopically expressing enteric gamma-actin

To determine the significance of actin isoforms in chemomechanical coupling, we compared tension and ATPase rate in heart myofilaments from nontransgenic (NTG) and transgenic (TG) mice in which enteric gamma-actin replaced >95% of the cardiac alpha-actin. Enteric gamma-actin was expressed against three backgrounds: mice expressing cardiac alpha-actin, heterozygous null cardiac alpha-actin mice, and homozygous null cardiac alpha-actin mice. There were no differences in maximum Ca(2+) activated tension or maximum rate of tension redevelopment after a quick release and rapid restretch protocol between TG and NTG skinned fiber bundles. However, compared with NTG controls, Ca(2+) sensitivity of tension was significantly decreased and economy of tension development was significantly increased in myofilaments from all TG hearts. Shifts in myosin isoform population could not fully account for this increase in the economy of force production of TG myofilaments. Our results indicate that an exchange of cardiac alpha-actin with an actin isoform differing in only five amino acids has a significant impact on both Ca(2+) regulation of cardiac myofilaments and the cross-bridge cycling rate.