Sarcomere Disarray Research Articles

Ventricular Myocyte Caspases Are Directly Responsible for Endotoxin-Induced Cardiac Dysfunction

Although most of the deleterious effects of sepsis-induced apoptosis have been attributed to increased lymphocyte cell death, caspase activation may directly alter cell function of different organ systems. We postulated that left ventricular (LV) cardiomyocyte caspase activation is directly involved in sepsis-induced heart contractile dysfunction. LV cardiomyocytes isolated 4 hours after rat treatment with endotoxin injection (10 mg/kg) displayed major reductions in contractile reserve and myofilament response to Ca2+. Concomitantly, endotoxin also induced increases in LV cardiomyocyte caspase-3, -8, and -9-like activities, which were associated with sarcomeric structure destruction and cleavage of components of the cardiac myofilament. Interestingly, zVAD.fmk treatment of septic rat prevented LV cardiomyocyte contractile dysfunction, reductions in myofilament response to calcium, troponin T cleavage, and sarcomere destruction. Serum (10%) of endotoxin-treated rats induced contractile dysfunction, caspase-3-like activity increase, and troponin T cleavage of naive LV cardiomyocytes. The effects of septic serum were prevented in LV cardiomyocytes isolated from zVAD.fmk- or zDEVD.cmk-treated rats or LV cardiomyocytes preincubated with zVAD.fmk or zDEVD.cmk. The results show an important relationship between endotoxin-induced caspase activation and reduced contractile reserve and sarcomere disarray at the level of single LV cardiomyocytes.

The structural examination of myocardial samples from patients with end-stage heart failure supported by ventricular assist devices using electron microscopy and amino acid analysis reveals low degree of reverse remodeling

Background Chronic heart failure is a multifactorial, progressive disease of many causes and is associated with complex ventricular remodeling. Deposition of extracellular matrix proteins and sarcomeric disarray of the myocytes occur in end-stage heart failure. Ventricular assist devices (VAD), implanted as bridge to transplantation, may reverse ventricular remodeling. Although successfully weaning patients from VAD support has been reported, it is not clear to what degree reversal of remodeling occurs in unloaded failing hearts. Because collagen deposition and ultrastructural disarray are hallmarks of myocardial remodeling, we analyzed the myocardial ultrastructure and collagen content of VAD-supported hearts before and after mechanical unloading. Methods We used amino acid analysis to measure collagen content (4-hydroxyproline content) in 24 transplant candidates receiving VAD support. We used transmission electron microscopy to examine the ultrastructure in 6 patients receiving VAD support. Results The 4-hydroxyproline content increased significantly at VAD implantation and was not altered by mechanical unloading. The ultrastructure showed signs of persisting cardiomyopathy. Conclusion Mechanical unloading does not alter the total collagen content of the supported, failing heart. Thus, structural reversal of the remodeling process associated with heart failure is not a general phenomenon in mechanically unloaded hearts.

Disruption of Caenorhabditis elegans Muscle Structure and Function Caused by Mutation of Troponin I

Caenorhabditis elegans strains mutant for the unc-27 gene show abnormal locomotion and muscle structure. Experiments revealed that unc-27 is one of four C. elegans troponin I genes and that three mutant alleles truncate the protein: recessive and presumed null allele e155 terminates after nine codons; semidominant su142sd eliminates the inhibitory and C-terminal regions; and semidominant su195sd abbreviates the extreme C-terminus. Assays of in vivo muscular performance at high and low loads indicated that su142sd is most deleterious, with e155 least and su195sd intermediate. Microscopy revealed in mutant muscle a prevalent disorder of dense body positioning and a less well defined sarcomeric structure, with small islands of thin filaments interspersed within the overlap region of A bands and even within the H zone. The mutants’ rigid paralysis and sarcomeric disarray are consistent with unregulated contraction of the sarcomeres, in which small portions of each myofibril shorten irregularly and independently of one another, thereby distorting the disposition of filaments. The exacerbated deficits of su142sd worms are compatible with involvement in vivo of the N-terminal portion of troponin I in enhancing force production, and the severe impairment associated with su195sd highlights importance of the extreme C-terminus in the protein’s inhibitory function.

Myocardial hypertrophy in transgenic mice overexpressing human interleukin 1α

Background: Interleukin (IL)-1 has profound effects on nonimmune cells and organs, including the heart. The effects of IL-1 on transgenic hearts have not yet been described. Methods and Results: We generated transgenic mice overexpressing the human IL-1 gene under control of the cytomegalovirus enhancer/chicken β-actin promoter. Heart weight–body weight ratio increased 1.4- to 2.2-fold in transgenic mice compared with wild-type mice. Lung weight–body weight ratio also increased in transgenic mice, all of which died within 14 days of birth. Light microscopy revealed concentric hypertrophy with cardiomyocyte hypertrophy in all transgenic mice and pulmonary edema in some of them. Electron microscopy showed myofilament loss and an increased number of giant mitochondria, but no sarcomere disarray. Northern blotting showed that gene expression had been reprogrammed in the left ventricle of transgenic mice. Expression of fetal-type genes such as prepro–atrial natriuretic factor and β-myosin heavy chain were increased, but voltage-dependent calcium channel messenger RNA expression was decreased in the left ventricle of transgenic mice compared with that of wild-type mice. Conclusions: IL-1 may cause structural and functional alterations in cardiac myocytes.

In vivo short-term expression of a hypertrophic cardiomyopathy mutation in adult rabbit myocardium: myofibrillar incorporation without early disarray.

Cardiac myocyte disarray is the pathological hallmark of hypertrophic cardiomyopathy (HCM), a disease of sarcomeric proteins. Mutations in the cardiac troponin T (cTnT), a major gene responsible for HCM, are associated with severe myocyte disarray. To study the pathogenesis of cardiac myocyte disarray, we expressed normal and mutant cTnT proteins in the myocardium of adult rabbits via direct intramyocardial injection of recombinant adenoviruses. Aliquots of 1010 plaque-forming units of normal (Ad/CMV/cTnT-Arg92) and mutant (Ad/CMV/cTnT-Gln92) recombinant viruses or a control vector (Ad/DeltaE) virus were mixed with equal aliquots of a reporter virus (Ad/CMV/Lac-Z) and co-injected into the myocardium of adult rabbits (n = 12). One week following gene transfer, thin myocardial sections were obtained and analyzed for beta-galactosidase, messenger RNA (mRNA) and protein expression, hematoxylin and eosin, Masson's trichrome, immunofluorescence staining, and electron microscopy. The efficiency of gene transfer varied from 2% to 60% of the cells in an area approximately 2.5 mm in length. Northern blotting confirmed expression of the transgenes into mRNA. Immunoblotting of the myofibrillar protein extracts and indirect immunofluorescence staining confirmed expression and incorporation of the transgene proteins into myofibrils. Expression of the mutant cTnT was up to 18% of the endogenous. Light and electron microscopic studies showed normal cardiac myocyte and sarcomere structures. Thus, despite incorporation of the mutant cTnT-Gln92, stable myofibrillar formation and sarcomere assembly proceeded in vivo. The absence of myocyte and sarcomere disarray may reflect the duration, or the level of expression, or the extent of myofibrillar incorporation of the mutant cTnT-Gln92, as well as the site and timing of expression of the transgenes, and interspecies variation in the pathogenesis of HCM.

Abstract

Familial hypertrophic cardiomyopathy (FHC) is a genetically and phenotypically heterogeneous disease with an autosomal dominant Mendelian inheritance. Mutations in four different genes coding for sarcomeric proteins have been identified to cause the disease. In 1990, a mutation in the β-myosin heavy chain (βMHC) gene was identified to cause familial hypertrophic cardiomyopathy. Soon after, more than 30 missense mutations and one deletion in the βMHC gene were identified to be responsible for FHC. This was followed by mutations in troponin T, α tropomyosin and cardiac myosin binding protein-C genes. In patients with familial hypertrophic cardiomyopathy due to βMHC mutations, the mutant βMHC mRNA and protein have been isolated from cardiac and skeletal muscle of these individuals. Functional studies have shown that the mutant βMHC protein has impaired actin-myosin interaction and expression of the mutant βMHC in feline cardiocytes disrupts the assembly of the sarcomere. Similarly, mutations in the other three genes lead to mutant proteins which interfere with the normal structure and function of the sarcomere, resulting in an identical phenotype of sarcomere disarray, the hallmark of FHC.

Molecular genetics of familial hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is a genetically and phenotypically heterogeneous disease with an autosomal dominant Mendelian inheritance. Mutations in four different genes coding for sarcomeric proteins have been identified to cause the disease. In 1990, a mutation in the β-myosin heavy chain (βMHC) gene was identified to cause familial hypertrophic cardiomyopathy. Soon after, more than 30 missense mutations and one deletion in the βMHC gene were identified to be responsible for FHC. This was followed by mutations in troponin T, α tropomyosin and cardiac myosin binding protein-C genes. In patients with familial hypertrophic cardiomyopathy due to βMHC mutations, the mutant βMHC mRNA and protein have been isolated from cardiac and skeletal muscle of these individuals. Functional studies have shown that the mutant βMHC protein has impaired actin-myosin interaction and expression of the mutant βMHC in feline cardiocytes disrupts the assembly of the sarcomere. Similarly, mutations in the other three genes lead to mutant proteins which interfere with the normal structure and function of the sarcomere, resulting in an identical phenotype of sarcomere disarray, the hallmark of FHC.

Expression of a mutation causing hypertrophic cardiomyopathy disrupts sarcomere assembly in adult feline cardiac myocytes.

Mutations in the beta-myosin heavy chain (beta MyHC) induce hypertrophic cardiomyopathy (HCM), cardiac hypertrophy, and sarcomere disarray, with the latter being the characteristic hallmark. Thus, we sought to determine whether expression of mutant beta MyHC in adult feline cardiac myocytes, a species known to develop HCM with a phenotype identical to that in humans, induces sarcomere disarray. A full-length beta MyHC cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg403Gln) was induced in the beta MyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant beta MyHC cDNAs were cloned into p delta E1spIB shuttle vector, downstream from a cytomegalovirus (CMV) promoter. Replication-deficient recombinant adenoviral constructs (Ad5/CMV/beta MyHC-N and Ad5/CMV/beta MyHC-403) were generated through homologous recombination of p delta E1spIB/CMV/beta MyHC-N or Ad5/CMV/beta MyHC-403 and pBHG10 after cotransfection in 293 host cells. Infection of COS-1 cells with the beta MyHC construct resulted in the expression of a full-length myosin protein. Efficiency of infection of isolated adult cardiac myocytes was > 95%. Expression of the beta MyHC constructs into mRNA at 48 hours after infection of feline cardiac myocytes was confirmed by reverse transcription-PCR. The net total protein and beta-myosin synthesis were determined by using the amount of incorporation of [3H]phenylalanine into total protein and beta-myosin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

407-5 Expression of a Mutation Causing Hypertrophic Cardiomyopathy Disrupts Sarcomere Assembly in Adult Feline Cardiac Myocytes

Mutations in the β myosin heavy chain (βMHC) gene cause hypertrophic cardiomyopathy (HCM), a disease characterized by sudden death, cardiac hypertrophy and sarcomere disarray. To determine the primary defect, we sought to express a mutant βMHC gene in adult feline cardiac myocytes. The feline model was selected since HCM is a common disease in cats with a phenotype identical to that of man and the cardiac myosin is βMHC as in man in contrastto αMHC in the mouse. A full length human βMHC cDNAwas cloned for the first time and an HCM causing mutation (Arg 403 Gln) was induced by site-directed mutagenesis. The adenoviral vector was selected for its high efficiency of gene transfer and reconstructed to accommodate the 7 kb βMHC cDNA and promoter. The normal and mutant βMHC cDNAs were cloned into a shuttle vector (ΔE1sp1 B) downstream to a cytomegalovirus promoter and the 7 kb fragment was incorporated through homologous recombination during co-transfection of pΔE1 sp1 B/CMV/βMHC and βBHG1 0 in 293 host cells. Adult feline cardiac myocytes were transfected with the recombinant viruses and examined at 48 and 120 hours after transfection. Expression of the βMHC into mRNA was confirmed by RT-PCR. Net myosin synthesis, determined by 3 H phenylalanine incorporation (48 hr), was greater in myocytes with normal or mutant βMHC constructs than control myocytes or those infected with vector alone (p < 0.05). Electron microscopy showed normal sarcomere assembly at 48 hours in >90% of myocytes, however, at 120 hours sarcomere disruption was evident in >50% of myocytes infected with mutant βMHC but remained intact in control myocytes and those infected with normal βMHC or vector alone (p = 003). In this study, we successfully incorporated a 7 kb βMHC/promoter construct into an adenoviral vector and showed its expression in adult feline cardiac myocytes. The mutant βMHC induced sarcomere and filament disarray similar to the lesion seen in man. This would indicate sarcomere disarray is the primary defect in HCM and the hypertrophy is compensatory.