Mutations in thin filament proteins that cause cardiomyopathy commonly cause an uncoupling of the relationship between the phosphorylation of troponin I and reduced Ca2+ sensitivity. Previously we showed that small molecules related to EGCG were able to restore the native response to mutant thin filaments in vitro and in MD simulations. However, 5,7-dimethoxychroman-3-yl 4-methoxybenzoate (compound 7) has an opposite effect-it causes mutant thin filament Ca2+ sensitivity to increase when cTroponin I is phosphorylated. In MD simulations of troponin with the TNNC1 G159D DCM mutation, we observed that compound 7 has unique effects upon troponin dynamics. Global parameters, such as interdomain hinge angle and Troponin C helix A/B angle distributions tend to be independent of phosphorylation unlike the phosphorylation-dependent changes observed with G159D alone or G159D plus recouplers such as silybin B. CCPTraj and Cluster Analysis suggest a novel preferred binding region between the extreme N terminus of cTroponin C and the switch peptide of cTroponin I.

Adrenergic activation of protein kinase A (PKA) targets the sarcolemma, sarcoplasmic reticulum, and contractile apparatus to increase contractile force and heart rate. In the thin filaments of the contractile apparatus, cTroponinI Ser22 and Ser23 are the targets for PKA phosphorylation. The effect of phosphorylation is a 2-3 fold decrease of affinity of cTn for Ca2+, associated with a higher rate of Ca2+ dissociation from cTnC leading to a faster relaxation rate of the cardiac muscle (lusitropy). This modulation of Ca2+-sensitivity is often suppressed by mutations that cause cardiomyopathy (uncoupling) and this could be sufficient to induce cardiomyopathy. Therefore, a drug that could restore the phosphorylation-dependent modulation of Ca2+-sensitivity could have potential for treatment of these pathologies. We have found that in single filament assays that a number of small molecules including silybin B, resveratrol and EGCG can restore coupling. We performed molecular dynamics simulations of the unphosphorylated and phosphorylated cardiac Troponin core with the TNNC1 G159D DCM mutation. We found that silybin B, EGCG and resveratrol restored the phosphoryation-induced change of most metrics to wild-type values, whilst silybin A, an inactive isomer of silybin B, and ECG did not. We analysed the atomic-level changes induced by ligand binding to explain recoupling. In parallel, we have extended our studies to intact TNNT2 R92Q-transfected cardiomyocytes. The mutation blunts the increased relaxation speed response to β1 adrenergic stimulation and we found that resveratrol, EGCG and silybin B could restore the β1 adrenergic response whilst silybin A did not. ### Competing Interest Statement The authors have declared no competing interest.

IntroductionAdrenergic activation of protein kinase A (PKA) in cardiac muscle targets the sarcolemma, sarcoplasmic reticulum, and contractile apparatus to increase contractile force and heart rate. In the thin filaments of the contractile apparatus, cardiac troponin I (cTnI) Ser22 and Ser23 in the cardiac-specific N-terminal peptide (NcTnI: residues 1 to 32) are the targets for PKA phosphorylation. Phosphorylation causes a 2-3 fold decrease of affinity of cTn for Ca2+ associated with a higher rate of Ca2+ dissociation from cTnC leading to a faster relaxation rate of the cardiac muscle (lusitropy). Cardiomyopathy-linked mutations primarily affect Ca2+ regulation or the PKA-dependent modulatory system, such that Ca2+-sensitivity becomes independent of phosphorylation level (uncoupling) and this could be sufficient to induce cardiomyopathy. A drug that could restore the phosphorylation-dependent modulation of Ca2+-sensitivity could have potential for treatment of these pathologies. We have found that a number of small molecules, including silybin B, resveratrol and EGCG, can restore coupling in single filament assays.MethodsWe did molecular dynamics simulations (5x1500ns for each condition) of the unphosphorylated and phosphorylated cardiac troponin core with the G159D DCM mutation in the presence of the 5 ligands and analysed the effects on several dynamic parameters. We also studied the effect of the ligands on the contractility of cardiac muscle myocytes with ACTC E99K and TNNT2 R92Q mutations in response to dobutamine.ResultsSilybin B, EGCG and resveratrol restored the phosphorylation-induced change in molecular dynamics to wild-type values, whilst silybin A, an inactive isomer of silybin B, and Epicatechin gallate, an EGCG analogue that does not recouple, did not. We analysed the atomic-level changes induced by ligand binding to explain recoupling. Mutations ACTC E99K and TNNT2 R92Q blunt the increased relaxation speed response to β1 adrenergic stimulation of cardiac myocytes and we found that resveratrol, EGCG and silybin B could restore the β1 adrenergic response, whereas silybin A did not.DiscussionThe uncoupling phenomenon caused by cardiomyopathy-related mutations and the ability of small molecules to restore coupling in vitro and lusitropy in myocytes is observed at the cellular, molecular and atomistic levels therefore, restoring lusitropy is a suitable target for treatment. Further research on compounds that restore lusitropy is thus indicated as treatments for genetic cardiomyopathies. Further molecular dynamics simulations could define the specific properties needed for recoupling and allow for the prediction and design of potential new drugs.

Human heart samples from the Sydney Heart Bank have become a de facto standard against which others can be measured. Crucially, the heart bank contains a lot of donor heart material: for most researchers this is the hardest to obtain and yet is necessary since we can only study the pathological human heart in comparison with a control, preferably a normal heart sample. It is not generally realised how important the control is for human heart studies. We review our studies on donor heart samples. We report the results obtained with 17 different donor samples collected from 1994 to 2011 and measured from 2005 to 2015 by our standard methodology for in vitro motility and troponin I phosphorylation measurements. The donor heart sample parameters are consistent between the hearts, over time and with different operators indicating that Sydney Heart Bank donor hearts are a valid baseline control for comparison with pathological heart samples. We also discuss to what extent donor heart samples are representative of the normal heart.

Hypertrophic cardiomyopathy (HCM) is a genetic form of left ventricular hypertrophy, primarily caused by mutations in sarcomere proteins. The cardiac remodeling that occurs as the disease develops can mask the pathogenic impact of the mutation. Here, to discriminate between mutation-induced and disease-related changes in myofilament function, we investigate the pathogenic mechanisms underlying HCM in a patient carrying a homozygous mutation (K280N) in the cardiac troponin T gene (TNNT2), which results in 100% mutant cardiac troponin T. We examine sarcomere mechanics and energetics in K280N-isolated myofibrils and demembranated muscle strips, before and after replacement of the endogenous troponin. We also compare these data to those of control preparations from donor hearts, aortic stenosis patients (LVHao), and HCM patients negative for sarcomeric protein mutations (HCMsmn). The rate constant of tension generation following maximal Ca2+ activation (k(ACT))and the rate constant of isometric relaxation (slow k(REL)) are markedly faster in K280N myofibrils than in all control groups. Simultaneous measurements of maximal isometric ATPase activity and Ca(2+)activated tension in demembranated muscle strips also demonstrate that the energy cost of tension generation is higher in the K280N than in all controls. Replacement of mutant protein by exchange with wild-type troponin in the K280N preparations reduces k(ACT), slow k(REL), and tension cost close to control values. In donor myofibrils and HCMsmn demembranated strips, replacement of endogenous troponin with troponin containing the K280N mutant increases k(ACT), slow k(REL), and tension cost. The K280N TNNT2 mutation directly alters the apparent cross-bridge kinetics and impairs sarcomere energetics. This result supports the hypothesis that inefficient ATP utilization by myofilaments plays a central role in the pathogenesis of the disease.

Mutations in contractile proteins can cause familial hypertrophic cardiomyopathy (HCM) or familial dilated cardiomyopathy (DCM). HCM has been linked to a higher myofilament Ca2+ sensitivity. In addition we have identified a molecular level dysfunction common to both HCM and DCM-causing mutations. This is an uncoupling of the relationship between troponin I (TnI) phosphorylation and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. Adrenergic response is blunted in vivo which predisposes to heart failure under stress. We have identified compounds that can specifically reverse these abnormalities in vitro and therefore have potential for treatment. Based on our lead compound, Epigallocatechin-3-Gallate (EGCG), we examined 40 compounds: variants of EGCG lacking the pyrogallol ring, variants of EGCG lacking the galloyl ring, silybin, its variants and stereoisomers and unrelated Hsp90 inhibitors and Ca2+-desensitisers. We found 23 compounds that reversed the uncoupling; many of these can re-couple independent of Ca2+-desensitization. 3 compounds desensitized but did not recouple, one compound has the reverse effect (P-TnI had higher Ca2+-sensitivity than unP TnI). We mapped EGCG, Silybin A and Silybin B binding to whole troponin by molecular dynamics simulations and found that they are usually located between the N-terminal phosphorylatable peptide of TnI and the N-terminal Ca2+ regulatory domain of TnC and differentially alter troponin dynamics. We have proposed a 4-state model to account for coupling, uncoupling and recoupling. We have established a biological assay platform for screening EGCG and related analogues in intact cardiomyocytes to study their effects on contractile regulation in vivo, using an E99K ACTC heterozygous-mutant HCM mouse model. Preliminary findings suggest that the response to dobutamine is blunted and that recoupling molecules appear to restore the adrenergic response.

Despite advancements in symptom management for heart failure (HF), this devastating clinical syndrome remains the leading cause of death in the developed world. Studies using animal models have greatly advanced our understanding of the molecular mechanisms underlying HF; however, differences in cardiac physiology and the manifestation of HF between animals, particularly rodents, and humans necessitates the direct interrogation of human heart tissue samples. Nevertheless, an ever-present concern when examining human heart tissue samples is the potential for artefactual changes related to temperature changes during tissue shipment or sample processing. Herein, we examined the effects of temperature on the post-translational modifications (PTMs) of sarcomeric proteins, the proteins responsible for muscle contraction, under conditions mimicking those that might occur during tissue shipment or sample processing. Using a powerful top-down proteomics method, we found that sarcomeric protein PTMs were differentially affected by temperature. Specifically, cardiac troponin I and enigma homolog isoform 2 showed robust increases in phosphorylation when tissue was incubated at either 4 °C or 22 °C. The observed increase is likely due to increased cyclic AMP levels and activation of protein kinase A in the tissue. On the contrary, cardiac troponin T and myosin regulatory light chain phosphorylation decreased when tissue was incubated at 4 °C or 22 °C. Furthermore, significant protein degradation was also observed after incubation at 4 °C or 22 °C. Overall, these results indicate that temperature exerts various effects on sarcomeric protein PTMs and careful tissue handling is critical for studies involving human heart samples. Moreover, these findings highlight the power of top-down proteomics for examining the integrity of cardiac tissue samples.

The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal Ca2+ regulation via troponin. HCM is usually linked to higher myofilament Ca2+-sensitivity whilst in both HCM and DCM mutant tissue there is often an uncoupling of the relationship between troponin I (TnI) phosphorylation by PKA and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heart to failure under stress. At present there are no compounds or interventions that can prevent or treat sarcomere cardiomyopathies. There is a need for novel therapies that act at a more fundamental level to affect the disease process. We demonstrated that epigallocatechin-3 gallate (EGCG) was found to be capable of restoring the coupled relationship between Ca2+-sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro, independent of the mutation (15 mutations tested). We have labeled this property “re-coupling.” The action of EGCG in vitro to reverse the abnormality caused by myopathic mutations would appear to be an ideal pharmaceutical profile for treatment of inherited HCM and DCM but EGCG is known to be promiscuous in vivo and is thus unsuitable as a therapeutic drug. We therefore investigated whether other structurally related compounds can re-couple myofilaments without these off-target effects. We used the quantitative in vitro motility assay to screen 40 compounds, related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca2+-sensitivity shift due to TnI phosphorylation was restored to 2.2 ± 0.01-fold (n = 19) compared to 2.0 ± 0.24-fold (n = 7) in wild-type thin filaments. Many of these compounds were either pure re-couplers or pure desensitizers, indicating these properties are independent; moreover, re-coupling ability could be lost with small changes of compound structure, indicating the possibility of specificity. Small molecules that can re-couple may have therapeutic potential. HIGHLIGHTS - Inherited cardiomyopathies are common diseases that are currently untreatable at a fundamental level and therefore finding a small molecule treatment is highly desirable. - We have identified a molecular level dysfunction common to nearly all mutations: uncoupling of the relationship between troponin I phosphorylation and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. - We have identified a new class of drugs that are capable of both reducing Ca2+-sensitivity and/or recouping the relationship between troponin I phosphorylation and Ca2+-sensitivity. - The re-coupling phenomenon can be explained on the basis of a single mechanism that is testable. - Measurements with a wide range of small molecules of varying structures can indicate the critical molecular features required for recoupling and allows the prediction of other potential re-couplers.

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Hypertrophic cardiomyopathy (HCM) is the most common single gene inherited cardiomyopathy. In cats (Felix catus) HCM is even more prevalent and affects 16% of the outbred population and up to 26% in pedigree breeds such as Maine Coon and Ragdoll. Homozygous MYBPC3 mutations have been identified in these breeds but the mutations in other cats are unknown. At the clinical and physiological level feline HCM is closely analogous to human HCM but little is known about the primary causative mechanism. Most identified HCM causing mutations are in the genes coding for proteins of the sarcomere. We therefore investigated contractile and regulatory proteins in left ventricular tissue from 25 cats, 18 diagnosed with HCM, including a Ragdoll cat with a homozygous MYBPC3 R820W, and 7 non-HCM cats in comparison with human HCM (from septal myectomy) and donor heart tissue. Myofibrillar protein expression was normal except that we observed 20–44% MyBP-C haploinsufficiency in 5 of the HCM cats. Troponin extracted from 8 HCM and 5 non-HCM cat hearts was incorporated into thin filaments and studied by in vitro motility assay. All HCM cat hearts had a higher (2.06 ± 0.13 fold) Ca2+-sensitivity than non-HCM cats and, in all the HCM cats, Ca2+-sensitivity was not modulated by troponin I phosphorylation. We were able to restore modulation of Ca2+-sensitivity by replacing troponin T with wild-type protein or by adding 100 μM Epigallocatechin 3-gallate (EGCG). These fundamental regulatory characteristics closely mimic those seen in human HCM indicating a common molecular mechanism that is independent of the causative mutation. Thus, the HCM cat is a potentially useful large animal model.

In both humans and mice, the Glu-99-Lys (E99K) mutation in the cardiac actin gene (ACTC) results in little understood apical hypertrophic cardiomyopathy (AHCM). To determine how cross-bridge kinetics change with AHCM development, we applied sinusoidal length perturbations to skinned papillary muscle fibres from 2- and 5-month old E99K transgenic (Tg) and non-transgenic (NTg) mice, and studied tension and its transients. These age groups were chosen because our preliminary studies indicated that AHCM develops with age. Fibres from 5-month old E99K mice showed significant decreases in tension, stiffness, the rate of the medium-speed exponential process and its magnitude compared to non-transgenic control. The nucleotide association constants increased with age, and they were significantly larger in E99K compared to NTg. However, there were no large differences in the rates of the cross-bridge detachment step, the rates of the force generation step, or the phosphate association constant. Our result on force/cross-bridge demonstrates that the decreased active tension of E99K fibres was caused by a decreased amount of force generated per each cross-bridge. The effects were generally less or insignificant at 2 months. A pCa-tension study showed increased Ca2+-sensitivity (pCa50) with age in both the E99K and NTg sample groups, and pCa50 was significantly larger (but only for 0.05–0.06 pCa units) in E99K than in NTg groups. A significant decrease in cooperativity (nH) was observed only in 5-month old E99K mice. We conclude that the AHCM-causing ACTC E99K mutation is associated with progressive alterations in biomechanical parameters, with changes smaller at 2 months but larger at 5 months, correlating with the development of AHCM.

Hypertrophic cardiomyopathy (HCM) is the most commonly inherited cardiomyopathy. In cats it is even more common: 15% of a large population of unselected outbred cats has HCM. HCM-causing mutations in sarcomeric proteins have been proposed to increase myofilament Ca2+-sensitivity but may have additional primary effects. We have previously found that in dilated cardiomyopathy (DCM)-causing mutations in thin filament proteins the relationship between phosphorylation at Ser 22 and 23 of cardiac troponin I (TnI) and Ca2+-sensitivity was abolished. This phenomenon has been termed ‘uncoupling’ and has been found for all thin-filament protein DCM-causing mutations investigated in vitro and in skinned myofibrils. It is associated with a blunted response to β1-adrenergic stimulation and reduced cardiac reserve that is potentially disease-causing. Interestingly, uncoupling might also be a characteristic of HCM. Uncoupling has been demonstrated in a number of HCM mutants in TnI and troponin T. To determine whether either of these phenomena is common to HCM mutations we have studied them using the in vitro motility assay. We have looked at mutations in TPM1 (E180G) and TNNT2 (Δ14, Δ28+7, R92L, R92Q, ΔE160, S179F, K273E and K280N) using recombinant and mutant cardiac troponin. All of these mutations showed uncoupling. The ACTC E99K mutation showed uncoupling in both isolated thin filaments and single myofibrils. Preliminary experiments were carried out with troponin from a Ragdoll cat heart with HCM due to the R820W mutation in MYBPC3 and a non-affected cat as control. The mutant thin filaments showed an increase in Ca2+-sensitivity and were also uncoupled. Thus, troponin from HCM cats was abnormal even though the mutation was in MyBP-C that was not present in the assay. Overall these results suggest the Ca2+-sensitising and uncoupling properties of HCM mutations may be more widely distributed than previously thought.

The only available crystal structure of the human cardiac troponin molecule (cTn) in the Ca(2+) activated state does not include crucial segments, including the N-terminus of the cTn inhibitory subunit (cTnI). We have applied all-atom molecular dynamics (MD) simulations to study the structure and dynamics of cTn, both in the unphosphorylated and bis-phosphorylated states at Ser23/Ser24 of cTnI. We performed multiple microsecond MD simulations of wild type (WT) cTn (6, 5 μs) and bisphosphorylated (SP23/SP24) cTn (9 μs) on a 419 amino acid cTn model containing human sequence cTnC (1-161), cTnI (1-171) and cTnT (212-298), including residues not present in the crystal structure. We have compared our results to previous computational studies, and proven that longer simulations and a water box of at least 25 Å are needed to sample the interesting conformational shifts both in the native and bis-phosphorylated states. As a consequence of the introduction into the model of the C-terminus of cTnT that was missing in previous studies, cTnC-cTnI interactions that are responsible for the cTn dynamics are altered. We have also shown that phosphorylation does not increase cTn fluctuations, and its effects on the protein-protein interaction profiles cannot be assessed in a significant way. Finally, we propose that phosphorylation could provoke a loss of Ca(2+) by stabilizing out-of-coordination distances of the cTnC's EF hand II residues, and in particular Ser 69.

The crystal structure of cTn does not include the crucial N-terminus cTnI region. We used all-atom Molecular Dynamics to understand the molecular events that follow phosphorylation in silico. We performed multiple microsecond MD simulations of wild type (WT) cTn (7250 ns so far) on a 419 amino acid cTn model containing human sequence cTnC (1-161), cTnI (1-172) and cTnT (216-298) including sequences not in the crystal structure. We have compared our results to previous computational studies, and proven that longer simulations and a water box of at least 25Å are needed to sample the interesting conformational shifts both in the native and phosphorylated states. As a consequence of the introduction into the model of the C-terminus of cTnT that was missing in previous studies TnC-TnI interactions that are responsible for the cTn dynamics are altered. We used cluster analysis to select 4 of our conformations at 750ns to start simulation of bisphosphorylated (TnI Ser23/24) cTn and have collected multiple 750ns simulations totaling 2650 νs so far. We show that phosphorylation does not have an appreciable effect on the cTn fluctuations and its flexible regions. The same molecular contacts between TnI 1-30, TnI 164-168 and TnC 1-88 and between TnI 1-40 and 138-147 and between TnC 1-41 and TnT 280-298 chare sampled in both phosphorylated and unphosphorylated simulations but phosphorylation changes the locations of interactions between the cTn subunits. We will describe ongoing simulations of Ca2+-cTn with HCM mutations TnI R145G and TnT K280N and DCM mutations TnI K36Q and TnC G159D in native and phosphorylated states.

In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomers are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. These phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients.

Patients with ‘stiff child’ syndrome usually have mutations at the interface of actin and tropomyosin that could affect the equilibrium of the Ca2+-dependent switch of muscle. ACTA1 K326N was previously reported and we now report two de novo TPM3 (Tpm3.12 protein) mutations, ΔE218 and ΔE224, resulting in a significant hypercontractile phenotype with marked congenital muscle stiffness associated with ventilatory failure in one case. The atomic resolution structure of tropomyosin bound to actin in the ‘switched off’ state shows that tropomyosin makes contact with actin at only two points: one of which is a cluster of basic amino acids: actin K326, K328 and R147. We have demonstrated that two Tpm2.2 mutations, ΔE139 and E181K, and the actin K326N mutation destabilize this actin-tropomyosin interface. We predicted that equivalent charge loss mutations at Tpm3.12 EE 218-219, EE 224-224, or ED 257-258 would also destabilise the interaction with actin leading to a partial switch-on of the muscle. The two newly discovered stiff patient mutation are located at two of the three predicted gain of function sites. We used the quantitative in vitro motility assay and skeletal muscle thin filaments containing recombinant mutant Tpm3.12 expressed in a Baculovirus/sf9 system. ΔE218 led to a 2.5-fold increase in Ca2+-sensitivity (EC50 ratio ΔE218/WT = 0.40 ± 0.07, ). ΔE224 also showed an increase in Ca2+-sensitivity by 2.2-fold (EC50 ratio ΔE224/WT for = 0.46 ± 0.09). It has been previously shown that there was a 2.5 fold increase in Ca2+ sensitivity for ACTC K326N mutation (EC50 ratio K326N/WT = 0.4 ± 0.05, p=0.07). The increased Ca2+-sensitivity indicates that both mutations cause a gain of function that was predicted from the structural analysis and that can account for the stiff patient syndrome.

We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca2+-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca2+-sensitivity when compared with donor heart troponin and the Ca2+-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca2+-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca2+-sensitivity and was also completely uncoupled. Five TnT mutations (Δ14, Δ28 + 7, ΔE160, S179F and K273E) studied in recombinant troponin increased Ca2+-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca2+-sensitisation together with uncoupling in vitro is the usual response and both factors may contribute to the HCM phenotype. We also found that Epigallocatechin-3-gallate (EGCG) can restore coupling to all uncoupled HCM-causing TnT mutations. In fact the combination of Ca2+-desensitisation and re-coupling due to EGCG completely reverses both the abnormalities found in troponin with a TnT HCM mutation suggesting it may have therapeutic potential.

Roughly half of all familial hypertrophic cardiomyopathy (HCM) cases can be linked to a sarcomere protein mutation, raising the possibility that the causative molecular pathways can be understood and corrected through pharmacotherapy. Nowhere is this more true than in the thin filament, for which all major components have been recombinantly expressed and extensively characterized by biophysical methods. It is generally accepted that for thin filament proteins, HCM-associated mutations increase the calcium sensitivity of the cardiac sarcomere. The prevailing theory is that increased calcium sensitivity causes hyper-activation of cardiac muscle, predisposing it to diastolic dysfunction and abnormal hypertrophy. Following similar reasoning, it has been postulated that familial dilated cardiomyopathy (DCM) mutations in the thin filament lead to decreased calcium sensitivity. However, decreased sensitivity has not been consistently demonstrated, causing some to seek alternative explanations. One interesting observation that has emerged in some studies is that thin filaments carrying DCM mutations are resistant to the desensitizing effect of protein kinase A (PKA) phosphorylation.1 In normal healthy individuals, sympathetic β-adrenergic stimulation drives PKA phosphorylation of cardiac troponin I Ser22/232,3 (among many other targets), and this speeds up sarcomeric calcium release and cardiac muscle relaxation. Messer and Marston have suggested that the lack of response to troponin I Ser22/23 phosphorylation, termed ‘uncoupling’, is a hallmark of familial DCM that also contributes to its pathogenesis. Could uncoupling be a feature central to HCM as well? Using in vitro motility assay (IVMA) to study mutations involving actin, tropomyosin, and the troponin complex, …