David F. Wieczorek

Mouse Model of a Familial Hypertrophic Cardiomyopathy Mutation in α-Tropomyosin Manifests Cardiac Dysfunction

To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant alpha-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the alpha-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous alpha-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain alpha-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca2+ sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC alpha-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.

Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos.

Tropomyosins (TMs) comprise a family of actin-binding proteins which play an important role in the regulation of contractility in muscle (cardiac, skeletal, and smooth) and nonmuscle cells. Although they are present in all cells, different isoforms are characteristic of specific cell types. In vertebrates, there are four different TM genes (alpha-TM, beta-TM, TM30, and TM4), three of which generate alternatively spliced isoforms. This study defines the expression patterns of these isoforms during murine embryogenesis, using both in vivo and in vitro conditions. The embryonic stem cell culture system, which has been shown to mimic different stages of mouse embryonic development, including the differentiation of primitive organ systems such as the myocardium, is used for our in vitro analysis. Our results demonstrate that several TM isoforms are expressed in specific developmental patterns, often correlated with the differentiation of particular tissues or organs. Surprisingly, other TMs, such as the striated muscle beta-TM and smooth muscle alpha-TM, are expressed constitutively. This study also demonstrates that there is an excellent correlation between the expression patterns of the TM isoforms observed in developing embryonic stem cells and mouse embryos. In addition, a quantitative molecular analysis of TM isoforms was conducted in embryonic, neonatal, and adult cardiac tissue. Our results show for the first time that the alpha- and beta-TM striated muscle transcripts are present in the earliest functional stages of the heart, and these TM isoforms are identical to those present throughout cardiac development.

Molecular and Physiological Effects of α-Tropomyosin Ablation in the Mouse

Abstract —Tropomyosin (TM) is an integral component of the thin filament in muscle fibers and is involved in regulating actin-myosin interactions. TM is encoded by a family of four alternatively spliced genes that display highly conserved nucleotide and amino acid sequences. To assess the functional and developmental significance of α-TM, the murine α-TM gene was disrupted by homologous recombination. Homozygous α-TM null mice are embryonic lethal, dying between 8 and 11.5 days post coitum. Mice that are heterozygous for α-TM are viable and reproduce normally. Heterozygous knockout mouse hearts show a 50% reduction in cardiac muscle α-TM mRNA, with no compensatory increase in transcript levels by striated muscle β-TM or TM-30 isoforms. Surprisingly, this reduction in α-TM mRNA levels in heterozygous mice is not reflected at the protein level, where normal amounts of striated muscle α-TM protein are produced and integrated in the myofibril. Quantification of α-TM mRNA bound in polysomal fractions reveals that both wild-type and heterozygous knockout animals have similar levels. These data suggest that a change in steady-state level of α-TM mRNA does not affect the relative amount of mRNA translated and amount of protein synthesized. Physiological analyses of myocardial and myofilament function show no differences between heterozygous α-TM mice and control mice. The present study suggests that translational regulation plays a major role in the control of TM expression.

Correlation Between Myofilament Response to Ca2+ and Altered Dynamics of Contraction and Relaxation in Transgenic Cardiac Cells That Express β-Tropomyosin

We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-beta-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-beta-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-beta-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (-log of [Ca2+]). Compared with controls, force generated by myofilaments from TG-beta-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca2+. However, the shift in pCa50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 microm was not significantly different between NTG and TG-beta-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-beta-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-beta-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.

EXCHANGE OF BETA - FOR ALPHA -TROPOMYOSIN IN HEARTS OF TRANSGENIC MICE INDUCES CHANGES IN THIN FILAMENT RESPONSE TO CA2+, STRONG CROSS-BRIDGE BINDING, AND PROTEIN PHOSPHORYLATION

Despite its potential as a key determinant of the functional state of striated muscle, the impact of tropomyosin (Tm) isoform switching on mammalian myofilament activation and regulation in the intact lattice remains unclear. Using a transgenic approach to specifically exchange β-Tm for the native α-Tm in mouse hearts, we have been able to uncover novel functions of Tm isoform switching in the heart. The myofilaments containing β-Tm demonstrated an increase in the activation of the thin filament by strongly bound cross-bridges, an increase in Ca sensitivity of steady state force, and a decrease in the rightward shift of the Ca-force relation induced by cAMP-dependent phosphorylation. Our results are the first to demonstrate the specific effects of Tm isoform switching on mammalian thin filament activation in the intact lattice and suggest an important role for Tm in modulation of myofilament activity by phosphorylation of troponin.

Dilated Cardiomyopathy Mutant Tropomyosin Mice Develop Cardiac Dysfunction With Significantly Decreased Fractional Shortening and Myofilament Calcium Sensitivity

Mutations in striated muscle &agr;-tropomyosin (&agr;-TM), an essential thin filament protein, cause both dilated cardiomyopathy (DCM) and familial hypertrophic cardiomyopathy. Two distinct point mutations within &agr;-tropomyosin are associated with the development of DCM in humans: Glu40Lys and Glu54Lys. To investigate the functional consequences of &agr;-TM mutations associated with DCM, we generated transgenic mice that express mutant &agr;-TM (Glu54Lys) in the adult heart. Results showed that an increase in transgenic protein expression led to a reciprocal decrease in endogenous &agr;-TM levels, with total myofilament TM protein levels remaining unaltered. Histological and morphological analyses revealed development of DCM with progression to heart failure and frequently death by 6 months. Echocardiographic analyses confirmed the dilated phenotype of the heart with a significant decrease in the left ventricular fractional shortening. Work-performing heart analyses showed significantly impaired systolic, and diastolic functions and the force measurements of cardiac myofibers revealed that the myofilaments had significantly decreased Ca2+ sensitivity and tension generation. Real-time RT-PCR quantification demonstrated an increased expression of &bgr;-myosin heavy chain, brain natriuretic peptide, and skeletal actin and a decreased expression of the Ca2+ handling proteins sarcoplasmic reticulum Ca2+-ATPase and ryanodine receptor. Furthermore, our study also indicates that the &agr;-TM54 mutation decreases tropomyosin flexibility, which may influence actin binding and myofilament Ca2+ sensitivity. The pathological and physiological phenotypes exhibited by these mice are consistent with those seen in human DCM and heart failure. As such, this is the first mouse model in which a mutation in a sarcomeric thin filament protein, specifically TM, leads to DCM.

Targeted Overexpression of Sarcolipin in the Mouse Heart Decreases Sarcoplasmic Reticulum Calcium Transport and Cardiac Contractility

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca2+ ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by β-adrenergic agonists.

Molecular and Functional Characterization of a Novel Cardiac-Specific Human Tropomyosin Isoform

Background— Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1&agr; (also called &agr;-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. Methods and Results— In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1&kgr;. By developing a TPM1&kgr;-specific antibody, we found that the TPM1&kgr; protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1&kgr; in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1&kgr;. Incorporation of increased levels of TPM1&kgr; protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1&kgr; compared with TPM1&agr;. Conclusions— This functional analysis of TPM1&kgr; provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.

A mouse model of familial hypertrophic cardiomyopathy caused by a α-tropomyosin mutation

Familial hypertrophic cardiomyopathy, a disease caused by mutations in cardiac contractile proteins, is characterized by left and/or right ventricular hypertrophy, myocyte disarray, fibrosis, and cardiac arrhythmias that may lead to premature sudden death. Five distinct point mutations within α-tropomyosin are associated with the development of familial hypertrophic cardiomyopathy. Two of these mutations are found within a troponin T binding site, located at amino acids 175 and 180. In this study, we analyze a transgenic mouse model for one of the mutations that occur at codon 180: a substitution of a glutamic acid for a glycine. These mice develop severe cardiac hypertrophy, substantial interstitial fibrosis, and have an increased heart weight/body weight ratio. Results show that calcium-handling proteins associated with the sarcoplasmic reticulum exhibit decreased expression. These alterations in gene expression, coupled with the structurally-altered tropomyosin, may contribute to the demonstrated decreased physiological performance exhibited by these transgenic mice. A DNA hybridization microarray analysis of the transgenic vs. control ventricular RNAs shows that 50 transcripts are differentially expressed by more than 100% during the onset of the hypertrophic process, many of which are associated with the extracellular matrix. This study demonstrates that mutations within tropomyosin can be severely disruptive of sarcomeric function, triggering a hypertrophic response coupled with a cascade of alterations in gene expression.

Ectopic expression of tropomyosin promotes myofibrillogenesis in mutant axolotl hearts

Expression of tropomyosin protein, an essential component of the thin filament, has been found to be drastically reduced in cardiac mutant hearts of the Mexican axolotl (Ambystoma mexicanum) with no formation of sarcomeric myofibrils. Therefore, this naturally occurring cardiac mutation is an appropriate model to examine the effects of delivering tropomyosin protein or tropomyosin cDNA into the deficient tissue. In this study, we describe the replacement of tropomyosin by using a cationic liposome transfection technique applied to whole hearts in vitro. When mouse α‐tropomyosin cDNA under the control of a cardiac‐specific α‐myosin heavy chain promoter was transfected into the mutant hearts, tropomyosin expression was enhanced resulting in the formation of well‐organized sarcomeric myofibrils. Transfection of a β‐tropomyosin construct under control of the same promoter did not result in enhanced organization of the myofibrils. Transfection of a β‐galactosidase reporter gene did not result in the formation of organized myofibrils or increased tropomyosin expression. These results demonstrate the importance of α‐tropomyosin to the phenotype of this mutation and to normal myofibril formation. Moreover, we have shown that a crucial contractile protein can be ectopically expressed in cardiac muscle that is deficient in this protein, with the resulting formation of organized sarcomeres. Dev. Dyn. 1998;213:412–420.