Evelyne M. Houang

Pathogenic peptide deviations support a model of adaptive evolution of chordate cardiac performance by troponin mutations

In cardiac muscle, the troponin (cTn) complex is a key regulator of myofilament calcium sensitivity because it serves as a molecular switch required for translating myocyte calcium fluxes into sarcomeric contraction and relaxation. Studies of several species suggest that ectotherm chordates have myofilaments with heightened calcium responsiveness. However, genetic polymorphisms in cTn that cause increased myofilament sensitivity to activating calcium in mammals result in cardiac disease including arrhythmias, diastolic dysfunction, and increased susceptibility to sudden cardiac death. We hypothesized that specific residue modifications in the regulatory arm of troponin I (TnI) were critical in mediating the observed decrease in myofilament calcium sensitivity within the mammalian taxa. We performed large-scale phylogenetic analysis, atomic resolution molecular dynamics simulations and modeling, and computational alanine scanning. This study provides evidence that a His to Ala substitution within mammalian cardiac TnI (cTnI) reduced the thermodynamic potential at the interface between cTnI and cardiac TnC (cTnC) in the calcium-saturated state by disrupting a strong intermolecular electrostatic interaction. This key residue modification reduced myofilament calcium sensitivity by making cTnI molecularly untethered from cTnC. To meet the requirements for refined mammalian adult cardiac performance, we propose that compensatory evolutionary pressures favored mutations that enhanced the relaxation properties of cTn by decreasing its sensitivity to activating calcium.

Membrane-stabilizing copolymers confer marked protection to dystrophic skeletal muscle in vivo.

Duchenne muscular dystrophy (DMD) is a fatal disease of striated muscle deterioration. A unique therapeutic approach for DMD is the use of synthetic membrane stabilizers to protect the fragile dystrophic sarcolemma against contraction-induced mechanical stress. Block copolymer-based membrane stabilizer poloxamer 188 (P188) has been shown to protect the dystrophic myocardium. In comparison, the ability of synthetic membrane stabilizers to protect fragile DMD skeletal muscles has been less clear. Because cardiac and skeletal muscles have distinct structural and functional features, including differences in the mechanism of activation, variance in sarcolemma phospholipid composition, and differences in the magnitude and types of forces generated, we speculated that optimized membrane stabilization could be inherently different. Our objective here is to use principles of pharmacodynamics to evaluate membrane stabilization therapy for DMD skeletal muscles. Results show a dramatic differential effect of membrane stabilization by optimization of pharmacodynamic-guided route of poloxamer delivery. Data show that subcutaneous P188 delivery, but not intravascular or intraperitoneal routes, conferred significant protection to dystrophic limb skeletal muscles undergoing mechanical stress in vivo. In addition, structure-function examination of synthetic membrane stabilizers further underscores the importance of copolymer composition, molecular weight, and dosage in optimization of poloxamer pharmacodynamics in vivo.

Chemical End Group Modified Diblock Copolymers Elucidate Anchor and Chain Mechanism of Membrane Stabilization

Block copolymers can be synthesized in an array of architectures and compositions to yield diverse chemical properties. The triblock copolymer Poloxamer 188 (P188), the family archetype, consisting of a hydrophobic poly(propylene oxide) core flanked by hydrophilic poly(ethylene oxide) chains, can stabilize cellular membranes during stress. However, little is known regarding the molecular basis of membrane interaction by copolymers in living organisms. By leveraging diblock architectural design, discrete end-group chemistry modifications can be tested. Here we show evidence of an anchor and chain mechanism of interaction wherein titrating poly(propylene oxide) block end group hydrophobicity directly dictates membrane interaction and stabilization. These findings, obtained in cells and animals in vivo, together with molecular dynamics simulations, provide new insights into copolymer-membrane interactions and establish the diblock copolymer molecular architecture as a valuable platform to inform copolymer-biological membrane interactions. These results have implications for membrane stabilizers in muscular dystrophy and for other biological applications involving damaged cell membranes.

Molecular Determinants of Cardiac Myocyte Performance as Conferred by Isoform-Specific TnI Residues

Troponin I (TnI) is the molecular switch of the sarcomere. Cardiac myocytes express two isoforms of TnI during development. The fetal heart expresses the slow skeletal TnI (ssTnI) isoform and shortly after birth ssTnI is completely and irreversibly replaced by the adult cardiac TnI (cTnI) isoform. These two isoforms have important functional differences; broadly, ssTnI is a positive inotrope, especially under acidic/hypoxic conditions, whereas cTnI facilitates faster relaxation performance. Evolutionary directed changes in cTnI sequence suggest cTnI evolved to favor relaxation performance in the mammalian heart. To investigate the mechanism, we focused on several notable TnI isoform and trans-species-specific residues located in TnIs helix 4 using structure/function and molecular dynamics analyses. Gene transduction of adult cardiac myocytes by cTnIs with specific helix 4 ssTnI substitutions, Q157R/A164H/E166V/H173N (QAEH), and A164H/H173N (AH), were investigated. cTnI QAEH is similar in these four residues to ssTnI and nonmammalian chordate cTnIs, whereas cTnI AH is similar to fish cTnI in these four residues. In comparison to mammalian cTnI, cTnI QAEH and cTnI AH showed increased contractility and slowed relaxation, which functionally mimicked ssTnI expressing myocytes. cTnI QAEH molecular dynamics simulations demonstrated altered intermolecular interactions between TnI helix 4 and cTnC helix A, specifically revealing a new, to our knowledge, electrostatic interaction between R171of cTnI and E15 of cTnC, which structurally phenocopied the ssTnI conformation. Free energy perturbation calculation of cTnC Ca(2+) binding for these conformations showed relative increased calcium binding for cTnI QAEH compared to cTnI. Taken together, to our knowledge, these new findings provide evidence that the evolutionary-directed coordinated acquisition of residues Q157, A164, E166, H173 facilitate enhanced relaxation performance in mammalian adult cardiac myocytes.

PEO–PPO Diblock Copolymers Protect Myoblasts from Hypo-Osmotic Stress In Vitro Dependent on Copolymer Size, Composition, and Architecture

Poloxamer 188, a triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), protects cellular membranes from various stresses. Though numerous block copolymer variants exist, evaluation of alternative architecture, composition, and size has been minimal. Herein, cultured murine myoblasts are exposed to the stresses of hypotonic shock and isotonic recovery, and membrane integrity was evaluated by quantifying release of lactate dehydrogenase. Comparative evaluation of a systematic set of PEO-PPO diblock and PEO-PPO-PEO triblock copolymers demonstrates that the diblock architecture can be protective in vitro. Short PPO blocks hinder protection with >9 PPO units needed for protection at 150 μM and >16 units needed at 14 μM. Addition of a tert-butyl end group enhances protection at reduced concentration. When the end group and PPO length are fixed, increasing the PEO length improves protection. This systematic evaluation establishes a new in vitro screening tool for evaluating membrane-sealing amphiphiles and provides mechanistic insight to guide future copolymer design for membrane stabilization in vivo.

All-Atom Molecular Dynamics-Based Analysis of Membrane-Stabilizing Copolymer Interactions with Lipid Bilayers Probed under Constant Surface Tensions

An all-atom phospholipid bilayer and triblock copolymer model was developed for molecular dynamics (MD) studies. These were performed to investigate the mechanism of interaction between membrane-stabilizing triblock copolymer P188 and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayers under applied lateral surface tension (γ) to model membrane mechanical stress. Results showed that P188 insertion is driven by the hydrophobic poly(propylene oxide) (PPO) core and dependent on bilayer area per lipid. Moreover, insertion of P188 increased the bilayers resistance to mechanical rupture, as observed by a significant increase in the absolute lateral pressure required to disrupt the bilayer. To further investigate the specific chemical features of P188 underlying membrane stabilizer function, a series of MD simulations with triblock copolymers of the same class as P188 but of varying chemical composition and sizes were performed. Results showed that triblock copolymer insertion into the lipid bilayer is dependent on overall copolymer hydrophobicity, with higher copolymer hydrophobicity requiring a reduced bilayer area per lipid ratio for insertion. Further analysis revealed that the effect of copolymer insertion on membrane mechanical integrity was also dependent on hydrophobicity. Here, P188 insertion significantly increased the absolute apparent lateral pressure required to rupture the POPC bilayer, thereby protecting the membrane against mechanical stress. In marked contrast, highly hydrophobic copolymers decreased the lateral pressure necessary for membrane rupture and thus rendering the membrane significantly more susceptible to mechanical stress. These new in silico findings align with recent experimental findings using synthetic lipid bilayers and in muscle cells in vitro and mouse models in vivo. Collectively, these data underscore the importance of PEO-PPO-PEO copolymer chemical composition in copolymer-based muscle membrane stabilization in vitro and in vivo. All-atom modeling with MD simulations holds promise for investigating novel copolymers with enhanced membrane interacting properties.

Muscle membrane integrity in Duchenne muscular dystrophy: recent advances in copolymer-based muscle membrane stabilizers

The scientific premise, design, and structure-function analysis of chemical-based muscle membrane stabilizing block copolymers are reviewed here for applications in striated muscle membrane injury. Synthetic block copolymers have a rich history and wide array of applications from industry to biology. Potential for discovery is enabled by a large chemical space for block copolymers, including modifications in block copolymer mass, composition, and molecular architecture. Collectively, this presents an impressive chemical landscape to leverage distinct structure-function outcomes. Of particular relevance to biology and medicine, stabilization of damaged phospholipid membranes using amphiphilic block copolymers, classified as poloxamers or pluronics, has been the subject of increasing scientific inquiry. This review focuses on implementing block copolymers to protect fragile muscle membranes against mechanical stress. The review highlights interventions in Duchenne muscular dystrophy, a fatal disease of progressive muscle deterioration owing to marked instability of the striated muscle membrane. Biophysical and chemical engineering advances are presented that delineate and expand upon current understanding of copolymer-lipid membrane interactions and the mechanism of stabilization. The studies presented here serve to underscore the utility of copolymer discovery leading toward the therapeutic application of block copolymers in Duchenne muscular dystrophy and potentially other biomedical applications in which membrane integrity is compromised.

TnI Structural Interface with the N-Terminal Lobe of TnC as a Determinant of Cardiac Contractility

The heterotrimeric cardiac troponin complex is a key regulator of contraction and plays an essential role in conferring Ca2+ sensitivity to the sarcomere. During ischemic injury, rapidly accumulating protons acidify the myoplasm, resulting in markedly reduced Ca2+ sensitivity of the sarcomere. Unlike the adult heart, sarcomeric Ca2+ sensitivity in fetal cardiac tissue is comparatively pH insensitive. Replacement of the adult cardiac troponin I (cTnI) isoform with the fetal troponin I (ssTnI) isoform renders adult cardiac contractile machinery relatively insensitive to acidification. Alignment and functional studies have determined histidine 132 of ssTnI to be the predominant source of this pH insensitivity. Substitution of histidine at the cognate position 164 in cTnI confers the same pH insensitivity to adult cardiac myocytes. An alanine at position 164 of cTnI is conserved in all mammals, with the exception of the platypus, which expresses a proline. Prolines are biophysically unique because of their innate conformational rigidity and helix-disrupting function. To provide deeper structure-function insight into the role of the TnC-TnI interface in determining contractility, we employed a live-cell approach alongside molecular dynamics simulations to ascertain the chemo-mechanical implications of the disrupted helix 4 of cTnI where position 164 exists. This important motif belongs to the critical switch region of cTnI. Substitution of a proline at position 164 of cTnI in adult rat cardiac myocytes causes increased contractility independent of alterations in the Ca2+ transient. Free-energy perturbation calculations of cTnC-Ca2+ binding indicate no difference in cTnC-Ca2+ affinity. Rather, we propose the enhanced contractility is derived from new salt bridge interactions between cTnI helix 4 and cTnC helix A, which are critical in determining pH sensitivity and contractility. Molecular dynamics simulations demonstrate that cTnI A164P structurally phenocopies ssTnI under baseline but not acidotic conditions. These findings highlight the evolutionarily directed role of the TnI-cTnC interface in determining cardiac contractility.