Jil C. Tardiff

Cardiac troponin T mutations result in allele-specific phenotypes in a mouse model for hypertrophic cardiomyopathy.

Multiple mutations in cardiac troponin T (cTnT) can cause familial hypertrophic cardiomyopathy (FHC). Patients with cTnT mutations generally exhibit mild or no ventricular hypertrophy, yet demonstrate a high frequency of early sudden death. To understand the functional basis of these phenotypes, we created transgenic mouse lines expressing 30%, 67%, and 92% of their total cTnT as a missense (R92Q) allele analogous to one found in FHC. Similar to a mouse FHC model expressing a truncated cTnT protein, the left ventricles of all R92Q lines are smaller than those of wild-type. In striking contrast to truncation mice, however, the R92Q hearts demonstrate significant induction of atrial natriuretic factor and beta-myosin heavy chain transcripts, interstitial fibrosis, and mitochondrial pathology. Isolated cardiac myocytes from R92Q mice have increased basal sarcomeric activation, impaired relaxation, and shorter sarcomere lengths. Isolated working heart data are consistent, showing hypercontractility and diastolic dysfunction, both of which are common findings in patients with FHC. These mice represent the first disease model to exhibit hypercontractility, as well as a unique model system for exploring the cellular pathogenesis of FHC. The distinct phenotypes of mice with different TnT alleles suggest that the clinical heterogeneity of FHC is at least partially due to allele-specific mechanisms.

A truncated cardiac troponin T molecule in transgenic mice suggests multiple cellular mechanisms for familial hypertrophic cardiomyopathy.

Mutations in multiple cardiac sarcomeric proteins including myosin heavy chain (MyHC) and cardiac troponin T (cTnT) cause a dominant genetic heart disease, familial hypertrophic cardiomyopathy (FHC). Patients with mutations in these two genes have quite distinct clinical characteristics. Those with MyHC mutations demonstrate more significant and uniform cardiac hypertrophy and a variable frequency of sudden death. Patients with cTnT mutations generally exhibit mild or no hypertrophy, but a high frequency of sudden death at an early age. To understand the basis for these distinctions and to study the pathogenesis of the disease, we have created transgenic mice expressing a truncated mouse cTnT allele analogous to one found in FHC patients. Mice expressing truncated cTnT at low (< 5%) levels develop cardiomyopathy and their hearts are significantly smaller (18-27%) than wild type. These animals also exhibit significant diastolic dysfunction and milder systolic dysfunction. Animals that express higher levels of transgene protein die within 24 h of birth. Transgenic mouse hearts demonstrate myocellular disarray and have a reduced number of cardiac myocytes that are smaller in size. These studies suggest that multiple cellular mechanisms result in the human disease, which is generally characterized by mild hypertrophy, but, also, frequent sudden death.

Sarcomeric Proteins and Familial Hypertrophic Cardiomyopathy: Linking Mutations in Structural Proteins to Complex Cardiovascular Phenotypes

Hypertrophic Cardiomyopathy (HCM) is a relatively common primary cardiac disorder defined as the presence of a hypertrophied left ventricle in the absence of any other diagnosed etiology. HCM is the most common cause of sudden cardiac death in young people which often occurs without precedent symptoms. The overall clinical phenotype of patients with HCM is broad, ranging from a complete lack of cardiovascular symptoms to exertional dyspnea, chest pain, and sudden death, often due to arrhythmias. To date, 270 independent mutations in nine sarcomeric protein genes have been linked to Familial Hypertrophic Cardiomyopathy (FHC), thus the clinical variability is matched by significant genetic heterogeneity. While the final clinical phenotype in patients with FHC is a result of multiple factors including modifier genes, environmental influences and genotype, initial screening studies had suggested that individual gene mutations could be linked to specific prognoses. Given that the sarcomeric genes linked to FHC encode proteins with known functions, a vast array of biochemical, biophysical and physiologic experimental approaches have been applied to elucidate the molecular mechanisms that underlie the pathogenesis of this complex cardiovascular disorder. In this review, to illustrate the basic relationship between protein dysfunction and disease pathogenesis we focus on representative gene mutations from each of the major structural components of the cardiac sarcomere: the thick filament (β MyHC), the thin filament (cTnT and Tm) and associated proteins (MyBP-C). The results of these studies will lead to a better understanding of FHC and eventually identify targets for therapeutic intervention.

Decreased energetics in murine hearts bearing the R92Q mutation in cardiac troponin T

The thin filament protein cardiac troponin T (cTnT) is an important regulator of myofilament activation. Here we report a significant change in cardiac energetics in transgenic mice bearing the missense mutation R92Q within the tropomyosin-binding domain of cTnT, a mutation associated with a clinically severe form of familial hypertrophic cardiomyopathy. This functional domain of cTnT has recently been shown to be a crucial modulator of contractile function despite the fact that it does not directly interact with the ATP hydrolysis site in the myosin head. Simultaneous measurements of cardiac energetics using 31P NMR spectroscopy and contractile performance of the intact beating heart revealed both a decrease in the free energy of ATP hydrolysis available to support contractile work and a marked inability to increase contractile performance upon acute inotropic challenge in hearts from R92Q mice. These results show that alterations in thin filament protein structure and function can lead to significant defects in myocardial energetics and contractile reserve.

Thin Filament Mutations: Developing an Integrative Approach to a Complex Disorder

Sixteen years ago, mutations in cardiac troponin (Tn)T and &agr;-tropomyosin were linked to familial hypertrophic cardiomyopathy, thus transforming the disorder from a disease of the &bgr;-myosin heavy chain to a disease of the cardiac sarcomere. From the outset, studies suggested that mutations in the regulatory thin filament caused a complex, heterogeneous pattern of ventricular remodeling with wide variations in clinical expression. To date, the clinical heterogeneity is well matched by an extensive array of nearly 100 independent mutations in all components of the cardiac thin filament. Significant advances in our understanding of the biophysics of myofilament activation, coupled to the emerging evidence that thin filament linked cardiomyopathies are progressive, suggests that a renewed focus on the most proximal events in both the molecular and clinical pathogenesis of the disease will be necessary to achieve the central goal of using genotype information to manage affected patients. In this review, we examine the existing biophysical and clinical evidence in support of a more proximal definition of thin filament cardiomyopathies. In addition, new high-resolution, integrated approaches are presented to help define the way forward as the field works toward developing a more robust link between genotype and phenotype in this complex disorder.

Cardiac troponin T mutations: correlation between the type of mutation and the nature of myofilament dysfunction in transgenic mice

1 The heterogenic nature of familial hypertrophic cardiomyopathy (FHC) in humans suggests a link between the type of mutation and the nature of patho‐physiological alterations in cardiac myocytes. Exactly how FHC‐associated mutations in cardiac troponin T (cTnT) lead to impaired cardiac function is unclear. 2 We measured steady‐state isometric force and ATPase activity in detergent‐skinned cardiac fibre bundles from three transgenic (TG) mouse hearts in which 50, 92 and 6 % of the native cTnT was replaced by the wild type (WT) cTnT, R92Q mutant cTnT (R92Q) and the C‐terminal deletion mutant of cTnT (cTnTDEL), respectively. 3 Normalized pCa‐tension relationships of R92Q and cTnTDEL fibres demonstrated a significant increase in sensitivity to Ca2+ at short (2.0 μm) and long (2.3 μm) sarcomere lengths (SL). At short SL, the pCa50 values, representing the midpoint of the pCa‐tension relationship, were 5.69 ± 0.01, 5.96 ± 0.01 and 5.81 ± 0.01 for WT, R92Q and cTnTDEL fibres, respectively. At long SL, the pCa50 values were 5.81 ± 0.01, 6.08 ± 0.01 and 5.95 ± 0.01 for WT, R92Q and cTnTDEL fibres, respectively. 4 The difference in pCa required for half‐maximal activation (ΔpCa50) at short and long SL was 0.12 ± 0.01 for the R92Q (92 %) TG fibres, which is significantly less than the previously reported ΔpCa50 value of 0.29 ± 0.02 for R92Q (67 %) TG fibres. 5 At short SL, Ca2+‐activated maximal tension in both R92Q and cTnTDEL fibres decreased significantly (24 and 21 %, respectively; P < 0.005), with no corresponding decrease in Ca2+‐activated maximal ATPase activity. Therefore, at short SL, the tension cost in R92Q and cTnTDEL fibres increased by 35 and 29 %, respectively (P < 0.001). 6 The fibre bundles reconstituted with the recombinant mutant cTnTDEL protein developed only 37 % of the Ca2+‐activated maximal force developed by recombinant WT cTnT reconstituted fibre bundles, with no apparent changes in Ca2+ sensitivity. 7 Our data indicate that an important mutation‐linked effect on cardiac function is the result of an inefficient use of ATP at the myofilament level. Furthermore, the extent of the mutation‐induced dysfunction depends not only on the nature of the mutation, but also on the concentration of the mutant protein in the sarcomere.

Mechanochemotransduction During Cardiomyocyte Contraction Is Mediated by Localized Nitric Oxide Signaling

Nitric oxide exposed to mechanical stress reveals the chemical cues involved in altering Ca2+ signals that lead to arrhythmias. Pulling Harder on the Heartstings To eject blood, a beating heart must contract against afterload, the buildup of mechanical tension in the left ventricle, which imposes mechanical stress. Calcium signaling increases in cardiomyocytes in a beating heart to enhance the strength of the muscular contraction to cope with afterload. However, this increase in calcium signaling can lead to arrhythmias. Jian et al. analyzed cardiomyocytes embedded in a gel matrix that imposed mechanical strain resembling afterload and found that nitric oxide generated near ryanodine receptors, a group of intracellular calcium channels, contributed to the afterload-induced increase in calcium signaling. These results identify potential therapeutic targets for treating various heart diseases that are caused by excessive mechanical stress or dysregulated Ca2+ signaling. Cardiomyocytes contract against a mechanical load during each heartbeat, and excessive mechanical stress leads to heart diseases. Using a cell-in-gel system that imposes an afterload during cardiomyocyte contraction, we found that nitric oxide synthase (NOS) was involved in transducing mechanical load to alter Ca2+ dynamics. In mouse ventricular myocytes, afterload increased the systolic Ca2+ transient, which enhanced contractility to counter mechanical load but also caused spontaneous Ca2+ sparks during diastole that could be arrhythmogenic. The increases in the Ca2+ transient and sparks were attributable to increased ryanodine receptor (RyR) sensitivity because the amount of Ca2+ in the sarcoplasmic reticulum load was unchanged. Either pharmacological inhibition or genetic deletion of nNOS (or NOS1), but not of eNOS (or NOS3), prevented afterload-induced Ca2+ sparks. This differential effect may arise from localized NO signaling, arising from the proximity of nNOS to RyR, as determined by super-resolution imaging. Ca2+-calmodulin–dependent protein kinase II (CaMKII) and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) also contributed to afterload-induced Ca2+ sparks. Cardiomyocytes from a mouse model of familial hypertrophic cardiomyopathy exhibited enhanced mechanotransduction and frequent arrhythmogenic Ca2+ sparks. Inhibiting nNOS and CaMKII, but not NOX2, in cardiomyocytes from this model eliminated the Ca2+ sparks, suggesting mechanotransduction activated nNOS and CaMKII independently from NOX2. Thus, our data identify nNOS, CaMKII, and NOX2 as key mediators in mechanochemotransduction during cardiac contraction, which provides new therapeutic targets for treating mechanical stress–induced Ca2+ dysregulation, arrhythmias, and cardiomyopathy.

Targets for Therapy in Sarcomeric Cardiomyopathies

To date, no compounds or interventions exist that treat or prevent sarcomeric cardiomyopathies. Established therapies currently improve the outcome, but novel therapies may be able to more fundamentally affect the disease process and course. Investigations of the pathomechanisms are generating molecular insights that can be useful for the design of novel specific drugs suitable for clinical use. As perturbations in the heart are stage-specific, proper timing of drug treatment is essential to prevent initiation and progression of cardiac disease in mutation carrier individuals. In this review, we emphasize potential novel therapies which may prevent, delay, or even reverse hypertrophic cardiomyopathy caused by sarcomeric gene mutations. These include corrections of genetic defects, altered sarcomere function, perturbations in intracellular ion homeostasis, and impaired myocardial energetics.

Temporal and mutation-specific alterations in Ca2+ homeostasis differentially determine the progression of cTnT-related cardiomyopathies in murine models.

Naturally occurring mutations in cardiac troponin T (cTnT) result in a clinical subset of familial hypertrophic cardiomyopathy. To determine the mechanistic links between thin-filament mutations and cardiovascular phenotypes, we have generated and characterized several transgenic mouse models carrying cTnT mutations. We address two central questions regarding the previously observed changes in myocellular mechanics and Ca(2+) homeostasis: 1) are they characteristic of all severe cTnT mutations, and 2) are they primary (early) or secondary (late) components of the myocellular response? Adult left ventricular myocytes were isolated from 2- and 6-mo-old transgenic mice carrying missense mutations at residue 92, flanking the TNT1 NH(2)-terminal tail domain. Results from R92L and R92W myocytes showed mutation-specific alterations in contraction and relaxation indexes at 2 mo with improvements by 6 mo. Alterations in Ca(2+) kinetics remained consistent with mechanical data in which R92L and R92W exhibited severe diastolic impairments at the early time point that improved with increasing age. A normal regulation of Ca(2+) kinetics in the context of an altered baseline cTnI phosphorylation suggested a pathogenic mechanism at the myofilament level taking precedence for R92L. The quantitation of Ca(2+)-handling proteins in R92W mice revealed a synergistic compensatory mechanism involving an increased Ser16 and Thr17 phosphorylation of phospholamban, contributing to the temporal onset of improved cellular mechanics and Ca(2+) homeostasis. Therefore, independent cTnT mutations in the TNT1 domain result in primary mutation-specific effects and a differential temporal onset of altered myocellular mechanics, Ca(2+) kinetics, and Ca(2+) homeostasis, complex mechanisms which may contribute to the clinical variability in cTnT-related familial hypertrophic cardiomyopathy mutations.

A model of calcium-activation of the cardiac thin filament

The cardiac thin filament regulates actomyosin interactions through calcium-dependent alterations in the dynamics of cardiac troponin and tropomyosin. Over the past several decades, many details of the structure and function of the cardiac thin filament and its components have been elucidated. We propose a dynamic, complete model of the thin filament that encompasses known structures of cardiac troponin, tropomyosin, and actin and show that it is able to capture key experimental findings. By performing molecular dynamics simulations under two conditions, one with calcium bound and the other without calcium bound to site II of cardiac troponin C (cTnC), we found that subtle changes in structure and protein contacts within cardiac troponin resulted in sweeping changes throughout the complex that alter tropomyosin (Tm) dynamics and cardiac troponin--actin interactions. Significant calcium-dependent changes in dynamics occur throughout the cardiac troponin complex, resulting from the combination of the following: structural changes in the N-lobe of cTnC at and adjacent to sites I and II and the link between them; secondary structural changes of the cardiac troponin I (cTnI) switch peptide, of the mobile domain, and in the vicinity of residue 25 of the N-terminus; secondary structural changes in the cardiac troponin T (cTnT) linker and Tm-binding regions; and small changes in cTnC-cTnI and cTnT-Tm contacts. As a result of these changes, we observe large changes in the dynamics of the following regions: the N-lobe of cTnC, the mobile domain of cTnI, the I-T arm, the cTnT linker, and overlapping Tm. Our model demonstrates a comprehensive mechanism for calcium activation of the cardiac thin filament consistent with previous, independent experimental findings. This model provides a valuable tool for research into the normal physiology of cardiac myofilaments and a template for studying cardiac thin filament mutations that cause human cardiomyopathies.