Elizabeth A. Brundage

AMP-activated Protein Kinase Phosphorylates Cardiac Troponin I at Ser-150 to Increase Myofilament Calcium Sensitivity and Blunt PKA-dependent Function

Background: Myofilament protein phosphorylation is central to cardiac muscle contractile regulation. Results: AMP-activated protein kinase (AMPK) phosphorylates troponin I (TnI) to increase cardiac contraction and blunt the effects of TnI protein kinase A phosphorylation. Conclusion: AMPK myofilament signaling represents a novel mechanism to regulate cardiac contraction. Significance: AMPK links metabolics to TnI regulation of cardiac muscle function and adrenergic regulation. AMP-activated protein kinase (AMPK) is an energy-sensing enzyme central to the regulation of metabolic homeostasis. In the heart AMPK is activated during cardiac stress-induced ATP depletion and functions to stimulate metabolic pathways that restore the AMP/ATP balance. Recently it was demonstrated that AMPK phosphorylates cardiac troponin I (cTnI) at Ser-150 in vitro. We sought to determine if the metabolic regulatory kinase AMPK phosphorylates cTnI at Ser-150 in vivo to alter cardiac contractile function directly at the level of the myofilament. Rabbit cardiac myofibrils separated by two-dimensional isoelectric focusing subjected to a Western blot with a cTnI phosphorylation-specific antibody demonstrates that cTnI is endogenously phosphorylated at Ser-150 in the heart. Treatment of myofibrils with the AMPK holoenzyme increased cTnI Ser-150 phosphorylation within the constraints of the muscle lattice. Compared with controls, cardiac fiber bundles exchanged with troponin containing cTnI pseudo-phosphorylated at Ser-150 demonstrate increased sensitivity of calcium-dependent force development, blunting of both PKA-dependent calcium desensitization, and PKA-dependent increases in length dependent activation. Thus, in addition to the defined role of AMPK as a cardiac metabolic energy gauge, these data demonstrate AMPK Ser-150 phosphorylation of cTnI directly links the regulation of cardiac metabolic demand to myofilament contractile energetics. Furthermore, the blunting effect of cTnI Ser-150 phosphorylation cross-talk can uncouple the effects of myofilament PKA-dependent phosphorylation from β-adrenergic signaling as a novel thin filament contractile regulatory signaling mechanism.

Ryanodine receptor phosphorylation by oxidized CaMKII contributes to the cardiotoxic effects of cardiac glycosides

AIMS Recent studies suggest that proarrhythmic effects of cardiac glycosides (CGs) on cardiomyocyte Ca(2+) handling involve generation of reactive oxygen species (ROS). However, the specific pathway(s) of ROS production and the subsequent downstream molecular events that mediate CG-dependent arrhythmogenesis remain to be defined. METHODS AND RESULTS We examined the effects of digitoxin (DGT) on Ca(2+) handling and ROS production in cardiomyocytes using a combination of pharmacological approaches and genetic mouse models. Myocytes isolated from mice deficient in NADPH oxidase type 2 (NOX2KO) and mice transgenically overexpressing mitochondrial superoxide dismutase displayed markedly increased tolerance to the proarrhythmic action of DGT as manifested by the inhibition of DGT-dependent ROS and spontaneous Ca(2+) waves (SCW). Additionally, DGT-induced mitochondrial membrane potential depolarization was abolished in NOX2KO cells. DGT-dependent ROS was suppressed by the inhibition of PI3K, PKC, and the mitochondrial KATP channel, suggesting roles for these proteins, respectively, in activation of NOX2 and in mitochondrial ROS generation. Western blot analysis revealed increased levels of oxidized CaMKII in WT but not in NOX2KO hearts treated with DGT. The DGT-induced increase in SCW frequency was abolished in myocytes isolated from mice in which the Ser 2814 CaMKII phosphorylation site on RyR2 is constitutively inactivated. CONCLUSION These results suggest that the arrhythmogenic adverse effects of CGs on Ca(2+) handling involve PI3K- and PKC-mediated stimulation of NOX2 and subsequent NOX2-dependent ROS release from the mitochondria; mitochondria-derived ROS then activate CaMKII with consequent phosphorylation of RyR2 at Ser 2814.

Combined troponin I Ser-150 and Ser-23/24 phosphorylation sustains thin filament Ca2 + sensitivity and accelerates deactivation in an acidic environment

The binding of Ca(2+) to troponin C (TnC) in the troponin complex is a critical step regulating the thin filament, the actin-myosin interaction and cardiac contraction. Phosphorylation of the troponin complex is a key regulatory mechanism to match cardiac contraction to demand. Here we demonstrate that phosphorylation of the troponin I (TnI) subunit is simultaneously increased at Ser-150 and Ser-23/24 during in vivo myocardial ischemia. Myocardial ischemia decreases intracellular pH resulting in depressed binding of Ca(2+) to TnC and impaired contraction. To determine the pathological relevance of these simultaneous TnI phosphorylations we measured individual TnI Ser-150 (S150D), Ser-23/24 (S23/24D) and combined (S23/24/150D) pseudo-phosphorylation effects on thin filament regulation at acidic pH similar to that in myocardial ischemia. Results demonstrate that while acidic pH decreased thin filament Ca(2+) binding to TnC regardless of TnI composition, TnI S150D attenuated this decrease rendering it similar to non-phosphorylated TnI at normal pH. The dissociation of Ca(2+) from TnC was unaltered by pH such that TnI S150D remained slow, S23/24D remained accelerated and the combined S23/24/150D remained accelerated. This effect of the combined TnI Ser-150 and Ser-23/24 pseudo-phosphorylations to maintain Ca(2+) binding while accelerating Ca(2+) dissociation represents the first post-translational modification of troponin by phosphorylation to both accelerate thin filament deactivation and maintain Ca(2+) sensitive activation. These data suggest that TnI Ser-150 phosphorylation induced attenuation of the pH-dependent decrease in Ca(2+) sensitivity and its combination with Ser-23/24 phosphorylation to maintain accelerated thin filament deactivation may impart an adaptive role to preserve contraction during acidic ischemia pH without slowing relaxation.

Cardiac troponin I tyrosine 26 phosphorylation decreases myofilament Ca2+ sensitivity and accelerates deactivation.

Troponin I (TnI), the inhibitory subunit of the troponin complex, can be phosphorylated as a key regulatory mechanism to alter the calcium regulation of contraction. Recent work has identified phosphorylation of TnI Tyr-26 in the human heart with unknown functional effects. We hypothesized that TnI Tyr-26N-terminal phosphorylation decreases calcium sensitivity of the thin filament, similar to the desensitizing effects of TnI Ser-23/24 phosphorylation. Our results demonstrate that Tyr-26 phosphorylation and pseudo-phosphorylation decrease calcium binding to troponin C (TnC) on the thin filament and calcium sensitivity of force development to a similar magnitude as TnI Ser-23/24 pseudo-phosphorylation. To investigate the effects of TnI Tyr-26 phosphorylation on myofilament deactivation, we measured the rate of calcium dissociation from TnC. Results demonstrate that filaments containing Tyr-26 pseudo-phosphorylated TnI accelerate the rate of calcium dissociation from TnC similar to that of TnI Ser-23/24. Finally, to assess functional integration of TnI Tyr-26 with Ser-23/24 phosphorylation, we generated recombinant TnI phospho-mimetic substitutions at all three residues. Our biochemical analyses demonstrated no additive effect on calcium sensitivity or calcium-sensitive force development imposed by Tyr-26 and Ser-23/24 phosphorylation integration. However, integration of Tyr-26 phosphorylation with pseudo-phosphorylated Ser-23/24 further accelerated thin filament deactivation. Our findings suggest that TnI Tyr-26 phosphorylation functions similarly to Ser-23/24N-terminal phosphorylation to decrease myofilament calcium sensitivity and accelerate myofilament relaxation. Furthermore, Tyr-26 phosphorylation can buffer the desensitization of Ser-23/24 phosphorylation while further accelerating thin filament deactivation. Therefore, the functional integration of TnI phosphorylation may be a common mechanism to modulate Ser-23/24 phosphorylation function.

Obligatory role of neuronal nitric oxide synthase in the heart's antioxidant adaptation with exercise

Excessive oxidative stress in the heart results in contractile dysfunction. While antioxidant therapies have been a disappointment clinically, exercise has shown beneficial results, in part by reducing oxidative stress. We have previously shown that neuronal nitric oxide synthase (nNOS) is essential for cardioprotective adaptations caused by exercise. We hypothesize that part of the cardioprotective role of nNOS is via the augmentation of the antioxidant defense with exercise by positively shifting the nitroso-redox balance. Our results show that nNOS is indispensable for the augmented anti-oxidant defense with exercise. Furthermore, exercise training of nNOS knockout mice resulted in a negative shift in the nitroso-redox balance resulting in contractile dysfunction. Remarkably, overexpressing nNOS (conditional cardiac-specific nNOS overexpression) was able to mimic exercise by increasing VO2max. This study demonstrates that exercise results in a positive shift in the nitroso-redox balance that is nNOS-dependent. Thus, targeting nNOS signaling may mimic the beneficial effects of exercise by combating oxidative stress and may be a viable treatment strategy for heart disease.

Myofilament Calcium Sensitivity: Mechanistic Insight into TnI Ser-23/24 and Ser-150 Phosphorylation Integration.

Troponin I (TnI) is a major regulator of cardiac muscle contraction and relaxation. During physiological and pathological stress, TnI is differentially phosphorylated at multiple residues through different signaling pathways to match cardiac function to demand. The combination of these TnI phosphorylations can exhibit an expected or unexpected functional integration, whereby the function of two phosphorylations are different than that predicted from the combined function of each individual phosphorylation alone. We have shown that TnI Ser-23/24 and Ser-150 phosphorylation exhibit functional integration and are simultaneously increased in response to cardiac stress. In the current study, we investigated the functional integration of TnI Ser-23/24 and Ser-150 to alter cardiac contraction. We hypothesized that Ser-23/24 and Ser-150 phosphorylation each utilize distinct molecular mechanisms to alter the TnI binding affinity within the thin filament. Mathematical modeling predicts that Ser-23/24 and Ser-150 phosphorylation affect different TnI affinities within the thin filament to distinctly alter the Ca2+-binding properties of troponin. Protein binding experiments validate this assertion by demonstrating pseudo-phosphorylated Ser-150 decreases the affinity of isolated TnI for actin, whereas Ser-23/24 pseudo-phosphorylation is not different from unphosphorylated. Thus, our data supports that TnI Ser-23/24 affects TnI-TnC binding, while Ser-150 phosphorylation alters TnI-actin binding. By measuring force development in troponin-exchanged skinned myocytes, we demonstrate that the Ca2+ sensitivity of force is directly related to the amount of phosphate present on TnI. Furthermore, we demonstrate that Ser-150 pseudo-phosphorylation blunts Ser-23/24-mediated decreased Ca2+-sensitive force development whether on the same or different TnI molecule. Therefore, TnI phosphorylations can integrate across troponins along the myofilament. These data demonstrate that TnI Ser-23/24 and Ser-150 phosphorylation regulates muscle contraction in part by modulating different TnI interactions in the thin filament and it is the combination of these differential mechanisms that provides understanding of their functional integration.