Yuanhua Cheng

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 KCa 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 (kREL,slow) and a decrease in its duration (tREL,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 Ca(2+) binding. We found that introducing two phosphomimic mutations into sites S23/S24 had no significant effect on the coordinating residues of Ca(2+) 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.

Effects of HCM cTnI Mutation R145G on Troponin Structure and Modulation by PKA Phosphorylation Elucidated by Molecular Dynamics Simulations

Cardiac troponin (cTn) is a key molecule in the regulation of human cardiac muscle contraction. The N-terminal cardiac-specific peptide of the inhibitory subunit of troponin, cTnI (cTnI(1-39)), is a target for phosphorylation by protein kinase A (PKA) during β-adrenergic stimulation. We recently presented evidence indicating that this peptide interacts with the inhibitory peptide (cTnl(137-147)) when S23 and S24 are phosphorylated. The inhibitory peptide is also the target of the point mutation cTnI-R145G, which is associated with hypertrophic cardiomyopathy (HCM), a disease associated with sudden death in apparently healthy young adults. It has been shown that both phosphorylation and this mutation alter the cTnC-cTnI (C-I) interaction, which plays a crucial role in modulating contractile activation. However, little is known about the molecular-level events underlying this modulation. Here, we computationally investigated the effects of the cTnI-R145G mutation on the dynamics of cTn, cTnC Ca(2+) handling, and the C-I interaction. Comparisons were made with the cTnI-R145G/S23D/S24D phosphomimic mutation, which has been used both experimentally and computationally to study the cTnI N-terminal specific effects of PKA phosphorylation. Additional comparisons between the phosphomimic mutations and the real phosphorylations were made. For this purpose, we ran triplicate 150 ns molecular dynamics simulations of cTnI-R145G Ca(2+)-bound cTnC(1-161)-cTnI(1-172)-cTnT(236-285), cTnI-R145G/S23D/S24D Ca(2+)-bound cTnC(1-161)-cTnI(1-172)-cTnT(236-285), and cTnI-R145G/PS23/PS24 Ca(2+)-bound cTnC(1-161)-cTnI(1-172)-cTnT(236-285), respectively. We found that the cTnI-R145G mutation did not impact the overall dynamics of cTn, but stabilized crucial Ca(2+)-coordinating interactions. However, the phosphomimic mutations increased overall cTn fluctuations and destabilized Ca(2+) coordination. Interestingly, cTnI-R145G blunted the intrasubunit interactions between the cTnI N-terminal extension and the cTnI inhibitory peptide, which have been suggested to play a crucial role in modulating troponin function during β-adrenergic stimulation. These findings offer a molecular-level explanation for how the HCM mutation cTnI-R145G reduces the modulation of cTn by phosphorylation of S23/S24 during β-adrenergic stimulation.

Contractile properties of developing human fetal cardiac muscle

The contractile properties of human fetal cardiac muscle have not been previously studied. Small‐scale approaches such as isolated myofibril and isolated contractile protein biomechanical assays allow study of activation and relaxation kinetics of human fetal cardiac muscle under well‐controlled conditions. We have examined the contractile properties of human fetal cardiac myofibrils and myosin across gestational age 59–134 days. Human fetal cardiac myofibrils have low force and slow kinetics of activation and relaxation that increase during the time period studied, and kinetic changes may result from structural maturation and changes in protein isoform expression. Understanding the time course of human fetal cardiac muscle structure and contractile maturation can provide a framework to study development of contractile dysfunction with disease and evaluate the maturation state of cultured stem cell‐derived cardiomyocytes.

2-Deoxy adenosine triphosphate improves contraction in human end-stage heart failure

We are developing a novel treatment for heart failure by increasing myocardial 2 deoxy-ATP (dATP). Our studies in rodent models have shown that substitution of dATP for adenosine triphosphate (ATP) as the energy substrate in vitro or elevation of dATP in vivo increases myocardial contraction and that small increases in the native dATP pool of heart muscle are sufficient to improve cardiac function. Here we report, for the first time, the effect of dATP on human adult cardiac muscle contraction. We measured the contractile properties of chemically-demembranated multicellular ventricular wall preparations and isolated myofibrils from human subjects with end-stage heart failure. Isometric force was increased at both saturating and physiologic Ca(2+) concentrations with dATP compared to ATP. This resulted in an increase in the Ca(2+) sensitivity of force (pCa50) by 0.06 pCa units. The rate of force redevelopment (ktr) in demembranated wall muscle was also increased, as was the rate of contractile activation (kACT) in isolated myofibrils, indicating increased cross-bridge binding and cycling compared with ATP in failing human myocardium. These data suggest that dATP could increase dP/dT and end systolic pressure in failing human myocardium. Importantly, even though the magnitude and rate of force development were increased, there was no increase in the time to 50% and 90% myofibril relaxation. These data, along with our previous studies in rodent models, show the promise of elevating myocardial dATP to enhance contraction and restore cardiac pump function. These data also support further pre-clinical evaluation of this new approach for treating heart failure.

Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties, and Modulation by Protein Kinase A (PKA)-mediated Phosphorylation

Background: R146G and R21C mutations in cardiac TnI are associated with hypertrophic cardiomyopathy. Results: Both mutations blunt PKA-mediated effects on weakening cTnI-cTnC interaction and accelerating myofibril relaxation. Conclusion: Both mutations result in hypercontraction and impaired relaxation, which may contribute to increased risk to traumatic heart failure. Significance: This study increases mechanistic understanding of how single amino acid mutations result in cardiac contractile dysfunction. Two hypertrophic cardiomyopathy-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory peptide and the cardiac-specific N terminus. We recently reported that these regions may interact when Ser-23/Ser-24 are phosphorylated, weakening the interaction of cTnI with cardiac TnC. Little is known about how these mutations influence the affinity of cardiac TnC for cTnI (KC-I) or contractile kinetics during β-adrenergic stimulation. Here, we tested how cTnIR146G or cTnIR21C influences contractile activation and relaxation and their response to protein kinase A (PKA). Both mutations significantly increased Ca2+ binding affinity to cTn (KCa) and KC-I. PKA phosphorylation resulted in a similar reduction of KCa for all complexes, but KC-I was reduced only with cTnIWT. cTnIWT, cTnIR146G, and cTnIR21C were complexed into cardiac troponin and exchanged into rat ventricular myofibrils, and contraction/relaxation kinetics were measured ± PKA phosphorylation. Maximal tension (Tmax) was maintained for cTnIR146G- and cTnIR21C-exchanged myofibrils, and Ca2+ sensitivity of tension (pCa50) was increased. PKA phosphorylation decreased pCa50 for cTnIWT-exchanged myofibrils but not for either mutation. PKA phosphorylation accelerated the early slow phase relaxation for cTnIWT myofibrils, especially at Ca2+ levels that the heart operates in vivo. Importantly, this effect was blunted for cTnIR146G- and cTnIR21C-exchanged myofibrils. Molecular dynamics simulations suggest both mutations inhibit formation of intra-subunit contacts between the N terminus and the inhibitory peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation. Together, our results suggest that cTnIR146G and cTnIR21C blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory peptide.

2-Deoxyadenosine triphosphate restores the contractile function of cardiac myofibril from adult dogs with naturally occurring dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a major type of heart failure resulting from loss of systolic function. Naturally occurring canine DCM is a widely accepted experimental paradigm for studying human DCM. 2-Deoxyadenosine triphosphate (dATP) can be used by myosin and is a superior energy substrate over ATP for cross-bridge formation and increased systolic function. The objective of this study was to evaluate the beneficial effect of dATP on contractile function of cardiac myofibrils from dogs with naturally occurring DCM. We measured actomyosin NTPase activity and contraction/relaxation properties of isolated myofibrils from nonfailing (NF) and DCM canine hearts. NTPase assays indicated replacement of ATP with dATP significantly increased myofilament activity in both NF and DCM samples. dATP significantly improved maximal tension of DCM myofibrils to the NF sample level. dATP also restored Ca(2+) sensitivity of tension that was reduced in DCM samples. Similarly, dATP increased the kinetics of contractile activation (kACT), with no impact on the rate of cross-bridge tension redevelopment (kTR). Thus, the activation kinetics (kACT/kTR) that were reduced in DCM samples were restored for dATP to NF sample levels. dATP had little effect on relaxation. The rate of early slow-phase relaxation was slightly reduced with dATP, but its duration was not, nor was the fast-phase relaxation or times to 50 and 90% relaxation. Our findings suggest that myosin utilization of dATP improves cardiac myofibril contractile properties of naturally occurring DCM canine samples, restoring them to NF levels, without compromising relaxation. This suggests elevation of cardiac dATP is a promising approach for the treatment of DCM.

Cardiac troponin structure-function and the influence of hypertrophic cardiomyopathy associated mutations on modulation of contractility

Cardiac troponin (cTn) acts as a pivotal regulator of muscle contraction and relaxation and is composed of three distinct subunits (cTnC: a highly conserved Ca(2+) binding subunit, cTnI: an actomyosin ATPase inhibitory subunit, and cTnT: a tropomyosin binding subunit). In this mini-review, we briefly summarize the structure-function relationship of cTn and its subunits, its modulation by PKA-mediated phosphorylation of cTnI, and what is known about how these properties are altered by hypertrophic cardiomyopathy (HCM) associated mutations of cTnI. This includes recent work using computational modeling approaches to understand the atomic-based structural level basis of disease-associated mutations. We propose a viewpoint that it is alteration of cTnC-cTnI interaction (rather than the Ca(2+) binding properties of cTn) per se that disrupt the ability of PKA-mediated phosphorylation at cTnI Ser-23/24 to alter contraction and relaxation in at least some HCM-associated mutations. The combination of state of the art biophysical approaches can provide new insight on the structure-function mechanisms of contractile dysfunction resulting cTnI mutations and exciting new avenues for the diagnosis, prevention, and even treatment of heart diseases.

Finally, We Can Relax: A New Generation of Muscle Models that Incorporate Sarcomere Compliance

Relaxation is an important property of muscle activity, but is much less studied or understood than contraction. For skeletal muscle, relaxation is important for motor control of movement, breathing, and posture. For cardiac muscle, relaxation is critically important for diastolic function, allowing effective filling with blood for pumping (systole), during normal activity but especially so when heart rate increases during activity or stress. In both skeletal and cardiac muscle, contraction and relaxation occur in networks of myofibrils organized in parallel bundles (Fig. 1 A).

11. Gene Therapy Mediated Increase in dATP Improves Cardiac Performance in Models of Systolic Heart Failure

Heart Failure results in more deaths and hospitalizations than almost any other single cause. We are exploring gene therapy treatment strategies that increase 2 deoxy-adenosine triphosphate (dATP) levels via cardiac specific expression of ribonucleotide reductase (RNR) in heart muscle. dATP is produced by RNR. In vitro studies show it increases the magnitude & rate of contraction in rodent, pig & failing human heart muscle without altering calcium handling or slowing relaxation. RNR expression in heart muscle increases intracellular dATP, cardiomyocyte contraction & cardiac performance in rodents. To do this we ligated a cDNA encoding both human RNR subunits to miniaturized cardiac specific enhancer/promoter portions of human cardiac troponin T and packaged the therapeutic “BB-R12” gene in AAV6. In the current study expanded the types of heart failure treated by BB-R12 by testing the effect of dATP on contractile function in a transgenic mouse model of familial dilated cardiomyopathy (DCM). These mice carry and phenocopy a missense mutation (D230N) in alpha-tropomyosin (Tm) that was identified in two DCM family cohorts that exhibit progressive ventricular dilation and loss of systolic function. We measured the effect of dATP on the Ca2+ sensitivity of force in demembranated trabeculae from young adult D230N & WT mice (3 mice & 8-10 trabeculae per group). pCa50 was decreased (lower Ca2+ sensitivity) for D230N Tm (5.47±0.01) compared to WT (5.59±0 0.03) mice, and substituting dATP for ATP rescued this defect (5.55±0.03). dATP also increased isometric force ~20% at pCa=5.8, the approximate level of intracellular Ca2+ seen in a cardiomyocyte twitch. We also measured force and the kinetics of activation & relaxation of isolated myofibrils. Force at pCa=5.6 decreased from 85±14 mN/mm2 in WT myofibrils to 52±7 mN/mm2 (p<0.05) in D230N Tm myofibrils. Importantly, the force deficit was improved to 72±14 mN/mm2 (p=0.47) with dATP. Similarly, the activation rate decreased in D230N Tm myofibrils (1.6±0.2 s-1) vs. WT mice (3.5±0.3 s-1), and this was partially corrected by dATP (2.5±0.4 s-1). dATP also corrected alterations in the early, slow relaxation phase of myofibrils from D230N Tm mice. In ongoing studies, we are systemically injecting 4-6 week old D230N Tm mice with AAV6-BB-R12 to determine how this affects cardiac function acutely, as well as the time course of reduced systolic function, progressive ventricular dilation & wall remodeling. In related studies, we found AAV6-BB-R12 rescues cardiac function in an infarcted pig heart failure model. These studies assess the ability of BB-R12 to acutely treat diseased hearts and act as a preventative agent in progressive heart failure.