Myocyte cytoskeletal disorganization and right heart failure in hypoxia-induced neonatal pulmonary hypertension.

Previous studies have demonstrated that environmentally or genetically induced changes in the intracellular proteins that compose the cytoskeleton can contribute to heart failure. Because neonatal right ventricular myocytes are immature and are in the process of significant cytoskeletal change, we hypothesized that they may be particularly susceptible to pressure stress. Newborn calves exposed to hypobaric hypoxia (barometric pressure = 430 mmHg) for 14 days developed severe pulmonary hypertension (pulmonary arterial pressure = 101 +/- 6 vs. 27 +/- 1 mmHg) and right heart failure compared with age-matched controls. Light microscopy showed partial loss of myocardial striations in the failing neonatal right but not left ventricles and in neither ventricle of adolescent cattle dying of altitude-induced right heart failure. In neonatal calves, immunohistochemical analysis of the cytoskeletal proteins (vinculin, metavinculin, desmin, vimentin, and cadherin) showed selectively, within the failing right ventricles, patchy areas characterized by loss and disorganization of costameres and intercalated discs. Within myocytes from the failing ventricles, vinculin and desmin were observed to redistribute diffusely within the cytosol, metavinculin appeared in disorganized clumps, and vimentin immunoreactivity was markedly decreased. Western blot analysis of the failing right ventricular myocardium showed, compared with control, vinculin and desmin to be little changed in total content but redistributed from insoluble (structural) to soluble (cytosolic) fractions; metavinculin total content was markedly decreased, tubulin content increased, particularly in the structural fraction, and cadherin total content and distribution were unchanged. We conclude that hypoxic pulmonary hypertensive-induced neonatal right ventricular failure is associated with disorganization of the cytoskeletal architecture.

Microtubules modulate cardiomyocyte beta-adrenergic response in cardiac hypertrophy.

The role of microtubules in modulating cardiomyocyte beta-adrenergic response was investigated in rats with cardiac hypertrophy. Male Sprague-Dawley rats underwent stenosis of the abdominal aorta (hypertensive, HT) or sham operation (normotensive, NT). Echocardiography and isolated left ventricular cardiomyocyte dimensions demonstrated cardiac hypertrophy in the HT rats after 30 wk. Cardiomyocyte microtubule fraction was assayed by high-speed centrifugation and Western blot. In contrast to previous reports of increased microtubules after acute pressure overload, microtubule fraction for HT was significantly lower than that for NT. Cardiomyocytes were exposed to either 1 microM colchicine, 10 microM taxol, or equivalent volume of vehicle. Colchicine decreased microtubules, and taxol increased microtubules in both groups. Cardiomyocyte cytosolic calcium ([Ca2+]c) and shortening/relaxation dynamics were assessed during exposure to increasing isoproterenol concentrations. The beta-adrenergic response for these variables in the HT group was blunted compared with NT. However, increased microtubule assembly by taxol partially recovered the normal beta-adrenergic response for time to peak [Ca2+]c, time to peak shortening, and mechanical relaxation variables. Microtubule assembly may play a significant role in determining cardiomyocyte beta-adrenergic response in chronic cardiac hypertrophy.

Coronary angioplasty for elderly patients with "high risk" unstable angina: short-term outcomes and long-term survival.

OBJECTIVES: We sought to compare the short- and long-term mortality rates in patients > or = 70 years old with unstable angina undergoing percutaneous transluminal coronary angioplasty (PTCA) with predicted coronary artery bypass graft surgery (CABG) short-term and U.S. census long-term mortality rates. BACKGROUND: Coronary angioplasty is an alternative revascularization strategy for patients with medically refractory rest angina and a high risk of adverse outcomes with CABG. Patients > or = 70 years old are a specific high risk subset. METHODS: A total of 131 consecutive patients aged > or = 70 years with unstable angina underwent PTCA; 82 (62%) of 131 had been refused CABG. Mortality over time was obtained from the Veterans Affairs Beneficiary Index Records Locator Subsystem. Predicted 30-day CABG-associated mortality was obtained from the Veterans Affairs Cardiac Risk Assessment Model. Mortality over time was expressed with Kaplan-Meier curves. RESULTS: The observed 30-day angioplasty survival rate was 87% compared with the predicted surgical 30-day survival rate of 85.5%. In those patients who survived 6 months after angioplasty (84%), their subsequent 1-, 2-, 3-, 4- and 5-year survival rates were comparable to age-matched subjects in the U.S. census. Mortality in certain subsets known to be at very high risk for CABG-for example, patients who had a previous CABG-was not high in this cohort of elderly subjects. The extremely high risk subsets identified in this PTCA cohort (shock, heart failure, pressors required, balloon pump required) were relatively infrequent subsets. CONCLUSIONS: For selected elderly patients with unstable angina deemed to be at "high risk" or even "prohibitive risk" for CABG, PTCA is an alternative revascularization strategy. The long-term mortality of successfully treated elderly patients is comparable to age-matched subjects. A prospective, multicenter, randomized trial of CABG versus PTCA, which includes patients > or = 70 years old, is being conducted (Veterans Affairs Cooperative Study 385: AWESOME).

Dilated cardiomyopathy associated with deficiency of the cytoskeletal protein metavinculin.

BACKGROUND: The cytoskeleton plays an important role in maintaining cell structure and integrity. Defects in cytoskeletal proteins can cripple cell strength and may cause cardiomyopathy. We analyzed heart tissues from subjects with dilated cardiomyopathy for abnormalities in the cardiac cytoskeleton. Metavinculin, a cardiac isoform of the cytoskeletal protein vinculin, connects actin microfilaments to the intercalated disk and membrane costameres of the heart. METHODS AND RESULTS: Metavinculin and vinculin transcripts and protein were analyzed by polymerase chain reaction (PCR) and Western blotting. Thirty-three human heart specimens were studied, including 5 normal controls, 4 subjects with ischemic cardiomyopathy, 1 with X-linked cardiomyopathy, and 23 with idiopathic dilated cardiomyopathy (IDC). PCR of cardiac cDNA detected absence of the metavinculin transcript in cardiac tissue from a subject with IDC. PCR of genomic DNA showed that the metavinculin exon was present but not utilized in the cardiac transcript. Western blot analysis demonstrated absence of metavinculin protein in the heart from this subject. Immunostaining of cardiac vinculin in this heart showed disorganized intercalated disk structures. Metavinculin deficiency was associated with normal cardiac expression of the cytoskeletal proteins vinculin, alpha-actinin, and dystrophin. Normal metavinculin expression in the other heart specimens suggests that the defect is specific in the IDC subject identified. CONCLUSIONS: These results demonstrate an association between metavinculin deficiency and dilated cardiomyopathy due to a defect in alternative mRNA splicing.

A 5' dystrophin duplication mutation causes membrane deficiency of alpha-dystroglycan in a family with X-linked cardiomyopathy.

5'-mutations in the dystrophin gene can result in cardiomyopathy without clinically-apparent skeletal myopathy. The effect of dystrophin mutations on the assembly and stability of the dystrophin associated protein (DAP) complex in human heart are not fully understood. The molecular defect in the dystrophin complex was explored in a family with an X-linked pedigree and severe dilated cardiomyopathy. Dystrophin gene analysis demonstrated a 5' duplication involving exons 2-7, which encodes the N-terminal actin binding domain of dystrophin. Ribonuclease protection and PCR assays demonstrated a reduction in muscle promoter transcribed dystrophin mRNA in the heart compared to skeletal muscle. A deficiency of cardiac dystrophin protein was observed by Western blot and lack of membrane localization by immunocytochemistry. The cardiac expression of the dystrophin related protein utrophin was increased, and the 43 kDa (beta-dystroglycan), 50 kDa (alpha-sarcoglycan) and 59 kDa (syntrophin) dystrophin associated proteins (DAPs) were co-isolated and present in nearly normal amounts in the membrane. However, cardiac dystrophin deficiency and increased utrophin expression were associated with loss of extracellular 156 kDa dystrophin associated glycoprotein (alpha-dystroglycan) binding to the cardiomyocyte membrane. alpha-Dystroglycan is responsible for linkage of the dystrophin complex to the extracellular matrix protein laminin. Therefore, 5' dystrophin mutations can reduce cardiac dystrophin mRNA, protein expression, and dystrophin function in X-linked cardiomyopathy (XLCM). The presence of membrane-associated beta-dystroglycan, alpha-sarcoglycan, syntrophin, and utrophin are insufficient to maintain cardiac function. This XLCM family has a 5' dystrophin gene mutation resulting in cardiac dystrophin deficiency and a loss of alpha-dystroglycan membrane binding.

Percutaneous transluminal angioplasty of internal mammary arteries in patients with rest angina.

Angioplasty of the internal mammary artery (IMA) bypass graft has been shown to be a safe and effective revascularization procedure. However, angiographic and long term clinical outcomes in the high-risk group of patients presenting with rest angina has not been well documented. We report the results of IMA angioplasty in 20 patients with rest angina out of 614 (3.2%) who received a left IMA graft at our institution between April 1987 and September 1994. All patients were admitted with rest angina, 12 patients demonstrated persistent ischemia despite medical therapy, two patients were in heart failure, and one patient was in cardiogenic shock. Balloon angioplasty was successful in 15 of 20 patients (75%). Failed angioplasty was associated with either severe IMA tortuousity (three patients) or inability to cross the anastomotic stenosis with the guide wire (two patients). Each of these five patients required angioplasty of either the native left anterior descending artery or other saphenous vein grafts for clinical stabilization. No patient suffered a major complication (myocardial infarction, emergent coronary bypass surgery, death). Clinical follow-up was obtained in all 20 patients (6 months, 7 years, mean 27 months). Twelve patients (60%) were asymptomatic or had stable angina at follow-up, and 8 returned with anginal symptoms. Four patients required repeat angioplasty for disease in other vessels, two were treated medically for angina, one underwent repeat CABG, and cardiac transplantation was performed in one patient for refractory heart failure. Angiographic follow up was obtained in 10/15 (66%) successful angioplasty patients, and only one patient demonstrated restenosis at the treated site (10%). During follow up one patient developed an IMA stenosis at a previous dissection site in the body of the graft that was treated with angioplasty. These results suggest that IMA angioplasty in patients with rest angina is associated with excellent long term patency and clinical efficacy, as well as low procedural risk.

Expression and regulation of the dystrophin Purkinje promoter in human skeletal muscle, heart, and brain.

Dystrophin mRNA transcripts from the P (Purkinje) promoter were shown to be differentially expressed in human skeletal muscle, heart, and brain. The expression pattern was characteristic of tissue type and developmental stage. Polymerase chain reaction (PCR) analysis of the P promoter transcripts in adult skeletal muscle and adult brain identified two alternatively spliced sequences, one that encodes a full-length dystrophin mRNA and a second that transcribes a termination codon 27 nucleotides (8 amino acids) after the ATG initiation site. Alternative splicing of this truncated coding transcript was developmentally regulated, and it was expressed as the major form in adult cortical brain and adult heart. The biological significance of this peptide remains unclear. The full-length transcript was the major form in fetal cortical brain and adult skeletal muscle. Ribonuclease protection assay demonstrated that as much as 20% of dystrophin transcription in normal adult skeletal muscle was derived from the full-length transcript from the P promoter. In contrast, adult heart did not express significant levels of P promoter derived transcripts. Thus, transcripts from the P promoter were found to be developmentally regulated in the brain, and its activity was differentially expressed in skeletal versus cardiac muscle tissues. These data show that the P promoter transcript displays a broader scope of expression, regulation, and complexity than previously appreciated.

Tumor necrosis factor-alpha and tumor necrosis factor receptors in the failing human heart.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that produces negative inotropic effects in the heart. Recently, elevated levels of TNF-alpha have been reported in patients with advanced congestive heart failure. Although TNF-alpha is thought to exert its deleterious effects by binding to two cell surface receptors, TNFR1 and TNFR2, the level of expression and regulation of TNF receptors in the heart in cardiac disease states is not known. METHODS AND RESULTS: We examined mRNA and protein levels for TNFR1, TNFR2, and TNF-alpha in explanted hearts from organ donors as well as in patients with end-stage dilated cardiomyopathy (DCM) and ischemic heart disease (IHD). Northern blot analysis revealed that mRNA for TNFR1 and TNFR2 was present in nonfailing, DCM, and IHD hearts. TNFR1 and TNFR2 receptor protein levels, as measured by ELISA, were decreased 60% in DCM and IHD patients compared with nonfailing hearts (P < .005). To determine a potential mechanism for the decrease in TNF receptor expression, we measured levels of circulating soluble TNF receptors (sTNFRs) in DCM and IHD patients. This analysis showed that there was a significant one-and-a-half to threefold increase in sTNFRs in DCM (P < .03) and IHD patients (P < .001). Another important finding was that TNF-alpha mRNA and TNF-alpha protein were present in the explanted hearts from DCM and IHD patients but not in nonfailing hearts. CONCLUSIONS: In summary, the results of this study constitute the initial demonstration that TNF receptor proteins are dynamically regulated in patients with advanced congestive heart failure. Moreover, the observation that failing hearts express elevated levels of TNF-alpha suggests that overexpression of this cytokine may be one of several different maladaptive mechanisms responsible for the progressive cardiac decompensation that occurs in advanced heart failure.

Expression and functional significance of tumor necrosis factor receptors in human myocardium.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine with potent negative inotropic properties, is elaborated in septic shock, acute myocarditis, reperfusion injury, and congestive heart failure. TNF-alpha acts by binding to two specific receptors: TNFR1 and TNFR2. However, neither the presence nor the significance of TNF receptors has been studied in the adult mammalian heart. METHODS AND RESULTS: In the present study, we showed that the adult heart expresses mRNA and receptor proteins for TNFR1 and TNFR2. Moreover, immunohistochemical staining studies localized TNFR1 and TNFR2 to the cardiac myocyte, providing a potential signaling pathway for the deleterious effects of TNF-alpha. The functional significance of the expression of TNFR1 and TNFR2 was explored with the use of a simple cell motion assay in which we assessed the effect(s) of TNF-alpha mutants known to bind selectively to human TNFR1 and TNFR2. We showed that the negative inotropic effect of wild-type TNF-alpha in isolated feline cardiac myocytes was mimicked by the TNF mutant that binds to TNFR1, whereas the TNF mutant that binds to TNFR2 had no significant effect on cell motion. CONCLUSIONS: Results of the present study show that the adult human heart expresses both mRNA and receptor proteins for TNFR1 and TNFR2; moreover, the negative inotropic effects of TNF-alpha in adult cardiac myocytes appear to be initiated by activation of TNFR1.

Identification, tissue-specific expression, and subcellular localization of the 80- and 71-kDa forms of myotonic dystrophy kinase protein.

The protein product of the myotonic dystrophy (DM) gene is a putative serine-threonine protein kinase (DM kinase). Previous reports have characterized the DM gene product as various 50-62-kDa proteins. The predicted protein size from DM cDNA sequence is 69 kDa. We therefore expressed a full-length recombinant human DM kinase protein and compared its size and expression to heart, cardiac Purkinje fibers, and skeletal muscle from normal and DM subjects. Recombinantly expressed DM kinase and endogenous DM kinase in human heart, displayed two immunoreactive DM kinase proteins with apparent molecular sizes of 71 and 80 kDa, suggesting that these prior reports are incorrect. In cardiac Purkinje fibers the 71-kDa protein was the major form, and in skeletal muscle the 80-kDa protein was the major form. Immunostaining showed DM kinase localized to neuromuscular junctions in skeletal muscle and intercalated discs in heart and Purkinje fibers. DM subjects showed low abundance of DM kinase in heart and skeletal muscle, suggesting haplotype insufficiency as a potential mechanism for disease expression. These studies describe differential expression of two protein forms of DM kinase, which are localized to specialized cellular structures associated with impulse transmission.

Mdx transgenic mouse: restoration of recombinant dystrophin to the dystrophic muscle.

We report the restoration of the 430-kD dystrophin in mdx, the mouse model of Duchenne muscular dystrophy, by expression of a single-copy recombinant dystrophin transgene. Muscle-specific expression was achieved using a creatine kinase promoter influenced by two enhancers. Immunostaining with anti-Xp21-coded dystrophin monoclonal antibodies showed that the recombinant dystrophin was localized to the muscle fiber membrane. However, there was variability in the level of dystrophin expression in various animals with aging, between fast and slow muscles, and within different regions of the same muscle. Curiously, recombinant dystrophin was relatively absent in the diaphragm muscle of these mdx transgenic animals. Our studies indicate that there is a direct correlation between the level of muscle fibers expressing recombinant dystrophin and the level of muscle fibers with peripheral nuclei, indicating an improvement in muscle pathology. These studies indicate that the regional expression of recombinant dystrophin in dystrophic muscle leads to regional restoration of normal muscle morphology.

An intact cysteine-rich domain is required for dystrophin function.

The carboxyl terminus of dystrophin is encoded by a highly conserved, alternatively spliced region of the gene. The few rare mutations reported in this region are of interest in unraveling the function of the dystrophin molecule. An unusual case of infantile onset Duchenne muscular dystrophy (DMD) with an internal 3' genomic deletion, and a membrane localized non-functional dystrophin protein, was used to explore the functional activity of this region. The patient's cDNA sequence showed an intragenic 1824-bp deletion precisely excising the cysteine rich and alternatively spliced COOH-terminal domains of dystrophin. The unaltered final 2.7 kb of the patients transcript was defined as a single exon localized to two genomic fragments, with the 5.9 kb HindIII fragment containing the stop codon. To understand the significance of deletions in this important region of the dystrophin gene, we mapped the order and cDNA coordinates for the 3' genomic HindIII fragments encoding the cysteine rich and alternative splicing domains. This 3' gene map was used to compare the clinical phenotype of the other reported COOH-terminal deletions in the literature. Our analysis concludes that the cysteine-rich domain confers an important function for the dystrophin protein.

Expression and localization of dystrophin in human cardiac Purkinje fibers.

BACKGROUND: Mutations in the dystrophin gene produce clinical manifestations of disease in heart, brain, and skeletal muscle in patients with Duchenne and Beckers muscular dystrophy (DMD/BMD). Conduction disturbances and heart block contribute to cardiac decompensation in these patients, which suggests an important role for dystrophia in the cardiac conduction system. We therefore examined the messenger RNA (mRNA) expression and protein localization of dystrophin in normal human cardiac Purkinje fibers. METHODS AND RESULTS: Polymerase chain reaction amplification of isolated Purkinje fiber complementary DNA identified several alternatively spliced mRNA transcripts encoding for carboxy-terminal isoforms of the dystrophin protein. The predominant mRNA transcript detected was a splice form previously detected in the brain. Antipeptide antibodies specific for a carboxy-terminal dystrophin sequence were used for Western blot analysis and immunocytochemical localization. These antisera detect approximately 400,000-d immunoreactive band or bands on Western blot in normal heart and Purkinje fibers but not in DMD heart. Immunocytochemical staining showed that dystrophin was localized to the membrane surface of the Purkinje fiber. CONCLUSIONS: These results suggest that dystrophin may be an important molecule for membrane function in the Purkinje conduction system of the heart and support the hypothesis that defective dystrophin expression contributes to the cardiac conduction disturbances seen in DMD/BMD:

Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development.

Dystrophin transcripts were shown to be alternatively spliced in a pattern characteristic of both tissue type and developmental stage. Multiple novel spliced forms of dystrophin mRNA were identified in murine brain tissue, skeletal and cardiac muscle, diaphragm, and human cardiac Purkinje fibers. The transcript diversity was greatest in adult, non-skeletal muscle tissues. Sequence analysis revealed that four tandem exons of the murine gene are differentially spliced in at least 11 separate patterns to generate distinct isoforms. Two of these forms were observed in all tissues examined, while several others were uniquely observed in cardiac muscle and brain. Cardiac Purkinje fibers express an isoform primarily observed in brain tissue. Several spliced transcripts were observed only in postnatal development. Differential utilization of a fifth exon results in two mRNA splice forms that encode separate embryonic and adult C-termini of dystrophin. Comparison of murine with human dystrophin mRNAs showed that similar isoform expression patterns exist across species. These observations suggest that functionally distinct isoforms of the dystrophin protein are expressed in separate tissues and at different stages of development. These isoforms may be of significance in understanding the various tissue-specific effects produced by dystrophin gene mutations in Duchenne and Becker muscular dystrophy patients.

Alu-primed polymerase chain reaction for regional assignment of 110 yeast artificial chromosome clones from the human X chromosome: identification of clones associated with a disease locus.

Over 400 yeast artificial chromosome (YAC) clones were isolated from the human X chromosome, and 110 of these were assigned to regions defined by chromosome translocation and deletion breakpoints. Polymerase chain reaction using Alu primers was applied to YAC clones in order to generate probes, to identify overlapping clones, and to derive "fingerprints" and sequence data directly from total yeast DNA. Several clones were identified in regions of medical interest. One set of three overlapping clones was found to cross a chromosomal translocation implicated in Lowe syndrome. The regional assignment of groups of YAC clones provides initiation points for further attempts to develop large cloned contiguous sequences, as well as material for investigation of regions involved in genetic diseases.

Antithrombin III deficiency: decreased synthesis of a biochemically normal molecule.

A 29-yr-old white female has suffered from recurrent venous thromboses over the last 12 yr. Plasma antithrombin III (AT-III) levels were 48% of normal by immunoelectrophoresis and 56% by chromogenic assay. Three of four siblings and the father had similar AT-III levels without associated venous thromboses. Heparin-Sepharose chromatography demonstrated normal behavior of the patient's AT-III. Her purified AT-III could not be distinguished from AT-III purified from a normal control either by SDS polyacrylamide gel electrophoresis or by crossed immunoelectrophoresis, and the heparin cofactor activity and the progressive antithrombin activity of both AT-III samples were identical. Turnover studies were made in the patient using her own purified AT-III labeled with 131I, (*I). The results did not differ significantly from studies made with autologous *I-AT-III in two normal control women. Her fractional breakdown rate of 0.54 total plasma AT-III per day compared with 0.45 and 0.52 in the controls. These studies indicate that the patient synthesizes a normal AT-III molecule at half normal rates.