Adding dimension to cellular mechanotransduction: Advances in biomedical engineering of multiaxial cell-stretch systems and their application to cardiovascular biomechanics and mechano-signaling.

Hollow organs (e.g. heart) experience pressure-induced mechanical wall stress sensed by molecular mechano-biosensors, including mechanosensitive ion channels, to translate into intracellular signaling. For direct mechanistic studies, stretch devices to apply defined extensions to cells adhered to elastomeric membranes have stimulated mechanotransduction research. However, most engineered systems only exploit unilateral cellular stretch. In addition, it is often taken for granted that stretch applied by hardware translates 1:1 to the cell membrane. However, the latter crucially depends on the tightness of the cell-substrate junction by focal adhesion complexes and is often not calibrated for. In the heart, (increased) hemodynamic volume/pressure load is associated with (increased) multiaxial wall tension, stretching individual cardiomyocytes in multiple directions. To adequately study cellular models of chronic organ distension on a cellular level, biomedical engineering faces challenges to implement multiaxial cell stretch systems that allow observing cell reactions to stretch during live-cell imaging, and to calibrate for hardware-to-cell membrane stretch translation. Here, we review mechanotransduction, cell stretch technologies from uni-to multiaxial designs in cardio-vascular research, and the importance of the stretch substrate-cell membrane junction. We also present new results using our IsoStretcher to demonstrate mechanosensitivity of Piezo1 in HEK293 cells and stretch-induced Ca2+ entry in 3D-hydrogel-embedded cardiomyocytes.

Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype.

The spondylocostal dysostoses (SCDs) are a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis. Previously, we had identified two genes that cause a subset of autosomal recessive forms of this disease: DLL3 (SCD1) and MESP2 (SCD2). These genes are important components of the Notch signaling pathway, which has multiple roles in development and disease. Here, we have used a candidate-gene approach to identify a mutation in a third Notch pathway gene, LUNATIC FRINGE (LFNG), in a family with autosomal recessive SCD. LFNG encodes a glycosyltransferase that modifies the Notch family of cell-surface receptors, a key step in the regulation of this signaling pathway. A missense mutation was identified in a highly conserved phenylalanine close to the active site of the enzyme. Functional analysis revealed that the mutant LFNG was not localized to the correct compartment of the cell, was unable to modulate Notch signaling in a cell-based assay, and was enzymatically inactive. This represents the first known mutation in the human LFNG gene and reinforces the hypothesis that proper regulation of the Notch signaling pathway is an absolute requirement for the correct patterning of the axial skeleton.

A polymorphic modifier gene alters the hypertrophic response in a murine model of familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disorder caused by mutationally altered dominant-acting sarcomere proteins, exhibits significant clinical heterogeneity. To determine whether genetic background could influence the expression of this disease, we studied a murine model for this human condition. Hypertrophic responses to the Arg403Gln missense mutation in a cardiac myosin heavy chain gene were compared in 129SvEv (inbred; designated 129SvEv- alpha MHC403/+) and Black Swiss (outbred; designated BSw- alpha MHC403/+) strains. At 30-50 weeks of age all 129SvEv- alpha MHC403/+ showed left ventricular hypertrophy, while left ventricular wall thickness was increased in only half of BSw- alpha MHC403/+ mice demonstrating that a polymorphic modifier gene can determine the hypertrophic response to this dominant-acting sarcomere protein mutation. Further analysis suggests that SJL/J mice bear a recessive allele of this modifier gene that prevents a hypertrophic response to the Arg403Gln missense mutation. We conclude that genetic modifiers in mice, and presumably in man, can alter the hypertrophic response to sarcomere protein gene missense mutations.

Cardiomyopathy in Irx4-deficient mice is preceded by abnormal ventricular gene expression.

To define the role of Irx4, a member of the Iroquois family of homeobox transcription factors in mammalian heart development and function, we disrupted the murine Irx4 gene. Cardiac morphology in Irx4-deficient mice (designated Irx4(Delta ex2/Delta ex2)) was normal during embryogenesis and in early postnatal life. Adult Irx4(Delta ex2/Delta ex2) mice developed a cardiomyopathy characterized by cardiac hypertrophy and impaired contractile function. Prior to the development of cardiomyopathy, Irx4(Delta ex2/Delta ex2) hearts had abnormal ventricular gene expression: Irx4-deficient embryos exhibited reduced ventricular expression of the basic helix-loop-helix transcription factor eHand (Hand1), increased Irx2 expression, and ventricular induction of an atrial chamber-specific transgene. In neonatal hearts, ventricular expression of atrial natriuretic factor and alpha-skeletal actin was markedly increased. Several weeks subsequent to these changes in embryonic and neonatal gene expression, increased expression of hypertrophic markers BNP and beta-myosin heavy chain accompanied adult-onset cardiac hypertrophy. Cardiac expression of Irx1, Irx2, and Irx5 may partially compensate for loss of Irx4 function. We conclude that Irx4 is not sufficient for ventricular chamber formation but is required for the establishment of some components of a ventricle-specific gene expression program. In the absence of genes under the control of Irx4, ventricular function deteriorates and cardiomyopathy ensues.

Comparison of two murine models of familial hypertrophic cardiomyopathy.

Although sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), individuals bearing a mutant cardiac myosin binding protein C (MyBP-C) gene usually have a better prognosis than individuals bearing beta-cardiac myosin heavy chain (MHC) gene mutations. Heterozygous mice bearing a cardiac MHC missense mutation (alphaMHC(403/+) or a cardiac MyBP-C mutation (MyBP-C(t/+)) were constructed as murine FHC models using homologous recombination in embryonic stem cells. We have compared cardiac structure and function of these mouse strains by several methods to further define mechanisms that determine the severity of FHC. Both strains demonstrated progressive left ventricular (LV) hypertrophy; however, by age 30 weeks, alphaMHC(403/+) mice demonstrated considerably more LV hypertrophy than MyBP-C(t/+) mice. In older heterozygous mice, hypertrophy continued to be more severe in the alphaMHC(403/+) mice than in the MyBP-C(t/+) mice. Consistent with this finding, hearts from 50-week-old alphaMHC(403/+) mice demonstrated increased expression of molecular markers of cardiac hypertrophy, but MyBP-C(t/+) hearts did not demonstrate expression of these molecular markers until the mice were >125 weeks old. Electrophysiological evaluation indicated that MyBP-C(t/+) mice are not as likely to have inducible ventricular tachycardia as alphaMHC(403/+) mice. In addition, cardiac function of alphaMHC(403/+) mice is significantly impaired before the development of LV hypertrophy, whereas cardiac function of MyBP-C(t/+) mice is not impaired even after the development of cardiac hypertrophy. Because these murine FHC models mimic their human counterparts, we propose that similar murine models will be useful for predicting the clinical consequences of other FHC-causing mutations. These data suggest that both electrophysiological and cardiac function studies may enable more definitive risk stratification in FHC patients.

An abnormal Ca(2+) response in mutant sarcomere protein-mediated familial hypertrophic cardiomyopathy.

Dominant-negative sarcomere protein gene mutations cause familial hypertrophic cardiomyopathy (FHC), a disease characterized by left-ventricular hypertrophy, angina, and dyspnea that can result in sudden death. We report here that a murine model of FHC bearing a cardiac myosin heavy-chain gene missense mutation (alphaMHC(403/+)), when treated with calcineurin inhibitors or a K(+)-channel agonist, developed accentuated hypertrophy, worsened histopathology, and was at risk for early death. Despite distinct pharmacologic targets, each agent augmented diastolic Ca(2+) concentrations in wild-type cardiac myocytes; alphaMHC(403/+) myocytes failed to respond. Pretreatment with a Ca(2+)-channel antagonist abrogated diastolic Ca(2+) changes in wild-type myocytes and prevented the exaggerated hypertrophic response of treated alphaMHC(403/+) mice. We conclude that FHC-causing sarcomere protein gene mutations cause abnormal Ca(2+) responses that initiate a hypertrophic response. These data define an important Ca(2+)-dependent step in the pathway by which mutant sarcomere proteins trigger myocyte growth and remodel the heart, provide definitive evidence that environment influences progression of FHC, and suggest a rational therapeutic approach to this prevalent human disease.

Dilated cardiomyopathy and sensorineural hearing loss: a heritable syndrome that maps to 6q23-24.

BACKGROUND: Dilated cardiomyopathy (DCM) and sensorineural hearing loss (SNHL) are prevalent disorders that occur alone or as components of complex multisystem syndromes. Multiple genetic loci have been identified that, when mutated, cause DCM or SNHL. However, the isolated coinheritance of these phenotypes has not been previously recognized. METHODS AND RESULTS: Clinical evaluations of 2 kindreds demonstrated autosomal-dominant transmission and age-related penetrance of both SNHL and DCM in the absence of other disorders. Moderate-to-severe hearing loss was evident by late adolescence, whereas ventricular dysfunction produced progressive congestive heart failure after the fourth decade. DNA samples from the larger kindred (29 individuals) were used to perform a genome-wide linkage study. Polymorphic loci on chromosome 6q23 to 24 were coinherited with the disease (maximum logarithm of odds score, 4.88 at locus D6S2411). The disease locus must lie within a 2.8 cM interval between loci D6S975 and D6S292, a location that overlaps an SNHL disease locus (DFNA10). However, DFNA10 does not cause cardiomyopathy. The epicardin gene, which encodes a transcription factor expressed in the myocardium and cochlea, was assessed as a candidate gene by nucleotide sequence analysis; no mutations were identified. CONCLUSIONS: A syndrome of juvenile-onset SNHL and adult-onset DCM is caused by a mutation at 6q23 to 24 (locus designated CMD1J). Recognition of this cardioauditory disorder allows for the identification of young adults at risk for serious heart disease, thereby enabling early intervention. Definition of the molecular cause of this syndrome may provide new information about important cell physiology common to both the ear and heart.

Missense mutations in the rod domain of the lamin A/C gene as causes of dilated cardiomyopathy and conduction-system disease.

BACKGROUND: Inherited mutations cause approximately 35 percent of cases of dilated cardiomyopathy; however, few genes associated with this disease have been identified. Previously, we located a gene defect that was responsible for autosomal dominant dilated cardiomyopathy and conduction-system disease on chromosome 1p1-q21, where nuclear-envelope proteins lamin A and lamin C are encoded by the LMNA (lamin A/C) gene. Mutations in the head or tail domain of this gene cause Emery-Dreifuss muscular dystrophy, a childhood-onset disease characterized by joint contractures and in some cases by abnormalities of cardiac conduction during adulthood. METHODS: We evaluated 11 families with autosomal dominant dilated cardiomyopathy and conduction-system disease. Sequences of the lamin A/C exons were determined in probands from each family, and variants were confirmed by restriction-enzyme digestion. The genotypes of the family members were ascertained. RESULTS: Five novel missense mutations were identified: four in the alpha-helical-rod domain of the lamin A/C gene, and one in the lamin C tail domain. Each mutation caused heritable, progressive conduction-system disease (sinus bradycardia, atrioventricular conduction block, or atrial arrhythmias) and dilated cardiomyopathy. Heart failure and sudden death occurred frequently within these families. No family members with mutations had either joint contractures or skeletal myopathy. Serum creatine kinase levels were normal in family members with mutations of the lamin rod but mildly elevated in some family members with a defect in the tail domain of lamin C. CONCLUSIONS: Genetic defects in distinct domains of the nuclear-envelope proteins lamin A and lamin C selectively cause dilated cardiomyopathy with conduction-system disease or autosomal dominant Emery-Dreifuss muscular dystrophy. Missense mutations in the rod domain of the lamin A/C gene provide a genetic cause for dilated cardiomyopathy and indicate that this intermediate filament protein has an important role in cardiac conduction and contractility.

Dilated cardiomyopathy in homozygous myosin-binding protein-C mutant mice.

To elucidate the role of cardiac myosin-binding protein-C (MyBP-C) in myocardial structure and function, we have produced mice expressing altered forms of this sarcomere protein. The engineered mutations encode truncated forms of MyBP-C in which the cardiac myosin heavy chain-binding and titin-binding domain has been replaced with novel amino acid residues. Analogous heterozygous defects in humans cause hypertrophic cardiomyopathy. Mice that are homozygous for the mutated MyBP-C alleles express less than 10% of truncated protein in M-bands of otherwise normal sarcomeres. Homozygous mice bearing mutated MyBP-C alleles are viable but exhibit neonatal onset of a progressive dilated cardiomyopathy with prominent histopathology of myocyte hypertrophy, myofibrillar disarray, fibrosis, and dystrophic calcification. Echocardiography of homozygous mutant mice showed left ventricular dilation and reduced contractile function at birth; myocardial hypertrophy increased as the animals matured. Left-ventricular pressure-volume analyses in adult homozygous mutant mice demonstrated depressed systolic contractility with diastolic dysfunction. These data revise our understanding of the role that MyBP-C plays in myofibrillogenesis during cardiac development and indicate the importance of this protein for long-term sarcomere function and normal cardiac morphology. We also propose that mice bearing homozygous familial hypertrophic cardiomyopathy-causing mutations may provide useful tools for predicting the severity of disease that these mutations will cause in humans.

Electrophysiologic characteristics of accessory atrioventricular connections in an inherited form of Wolff-Parkinson-White syndrome.

INTRODUCTION: A familial form of Wolff-Parkinson-White syndrome (WPW) occurs in association with hypertrophic cardiomyopathy and intraventricular conduction abnormalities. This syndrome, demonstrating autosomal dominant inheritance and segregating with a high degree of penetrance but variable expressivity, has been genetically linked to chromosome 7q3. The purpose of this study is to detail the electrophysiologic characteristics of accessory atrioventricular connections (AC) in four members of a kindred with this syndrome. METHODS AND RESULTS: We clinically evaluated 32 members of a single kindred and identified 20 individuals with ventricular preexcitation, abnormal intraventricular conduction including complete AV block and/or ventricular hypertrophy. Genetic linkage analysis mapped the disease gene in this kindred to the chromosome 7q3 locus (maximum logarithm of the odds score = 6.88, theta = 0); recombination events in affected individuals reduced the genetic interval from 7 centimorgans (cM) to 5 cM. Electrophysiologic study of four individuals with preexcitation, identified seven AC (1 right sided, 3 septal, and 3 left sided). All four individuals had inducible orthodromic tachycardia; while three had multiple AC. Bidirectional conduction was demonstrated in 6 of 7 AC. Successful ablation was accomplished in 5 of 7 AC. CONCLUSION: The electrophysiologic characteristics and location of AC in family members having this complex cardiac phenotype are similar to those seen in individuals with isolated WPW. Identification of WPW in more than one family member should prompt clinical evaluation of relatives for additional findings of ventricular hypertrophy or conduction abnormalities.

Familial dilated cardiomyopathy locus maps to chromosome 2q31.

BACKGROUND: Inherited gene defects are an important cause of dilated cardiomyopathy. Although the chromosome locations of some defects and 1 disease gene (actin) have been identified, the genetic etiologies of most cases of familial dilated cardiomyopathy remain unknown. METHODS AND RESULTS: We clinically evaluated 3 generations of a kindred with autosomal dominant transmission of dilated cardiomyopathy. Nine surviving and affected individuals had early-onset disease (ventricular chamber dilation during the teenage years and congestive heart failure during the third decade of life). The disease was nonpenetrant in 2 obligate carriers. To identify the causal gene defect, linkage studies were performed. A new dilated cardiomyopathy locus was identified on chromosome 2 between loci GCG and D2S72 (maximum logarithm of odds [LOD] score=4.86 at theta=0). Because the massive gene encoding titin, a cytoskeletal muscle protein, resides in this disease interval, sequences encoding 900 amino acid residues of the cardiac-specific (N2-B) domain were analyzed. Five sequence variants were identified, but none segregated with disease in this family. CONCLUSIONS: A dilated cardiomyopathy locus (designated CMD1G) is located on chromosome 2q31 and causes early-onset congestive heart failure. Although titin remains an intriguing candidate gene for this disorder, a disease-causing mutation is not present in its cardiac-specific N2-B domain.

Neonatal cardiomyopathy in mice homozygous for the Arg403Gln mutation in the alpha cardiac myosin heavy chain gene.

Heterozygous mice bearing an Arg403Gln missense mutation in the alpha cardiac myosin heavy chain gene (alpha-MHC403/+) exhibit the histopathologic features of human familial hypertrophic cardiomyopathy. Surprisingly, homozygous alpha-MHC403/403 mice die by postnatal day 8. Here we report that neonatal lethality is caused by a fulminant dilated cardiomyopathy characterized by myocyte dysfunction and loss. Heart tissues from neonatal wild-type and alpha-MHC403/403 mice demonstrate equivalent switching of MHC isoforms; alpha isoforms in each increase from 30% at birth to 70% by day 6. Cardiac dimensions and function, studied for the first time in neonatal mice by high frequency (45 MHz) echocardiography, were normal at birth. Between days 4 and 6, alpha-MHC403/403 mice developed a rapidly progressive cardiomyopathy with left ventricular dilation, wall thinning, and reduced systolic contraction. Histopathology revealed myocardial necrosis with dystrophic calcification. Electron microscopy showed normal architecture intermixed with focal myofibrillar disarray. We conclude that 45-MHz echocardiography is an excellent tool for assessing cardiac physiology in neonatal mice and that the concentration of Gln403 alpha cardiac MHC in myocytes influences both cell function and cell viability. We speculate that variable incorporation of mutant and normal MHC into sarcomeres of heterozygotes may account for focal myocyte death in familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy and atrial fibrillation caused by Arg663His beta-cardiac myosin heavy chain mutation.

More than 40 different beta-cardiac myosin heavy chain (beta-MHC) missense mutations have been identified that cause familial hypertrophic cardiomyopathy (FHC). Some of these are recognized to have important clinical manifestations, such as an increased incidence of sudden death. We report that the beta-MHC missense mutation Arg663His causes predominant cardiac morphology and atrial fibrillation. Longitudinal clinical evaluations were performed in a kindred with FHC. The nucleotide sequence of the beta-MHC gene was analyzed to define the causal mutation. A missense mutation in the beta-MHC gene, Arg663His, was identified in 24 individuals. Clinical studies demonstrated modest left ventricular hypertrophy in affected individuals, predominantly localized in the proximal segment of the interventricular septum, which increased (average = 40 +/- 8%) during 7 years of follow-up. Results showed that 47% of Arg663His adults (age > 16 years) with ventricular hypertrophy developed atrial fibrillation, significantly more (p <0.001) than observed in ungenotyped FHC populations. Survival of affected individuals remained near normal. The beta-MHC missense mutation Arg663His causes a characteristic pattern of ventricular hypertrophy. Arg663His individuals have a markedly higher prevalence of atrial fibrillation, compared with a population with ungenotyped hypertrophic cardiomyopathy. The demonstration of phenotype as a direct consequence of genotype further extends the utility of molecular data in clinical medicine. Early identification of Arg663His individuals has the potential to minimize the serious sequelae of this arrhythmia in this FHC group.

Reduced penetrance, variable expressivity, and genetic heterogeneity of familial atrial septal defects.

BACKGROUND: Secundum atrial septal defect (ASD) is a common congenital heart malformation that occurs as an isolated anomaly in 10% of individuals with congenital heart disease. Although some embryological pathways have been elucidated, the molecular etiologies of ASD are not fully understood. Most cases of ASD are isolated, but some individuals with ASD have a family history of this defect or other congenital heart malformations. METHODS AND RESULTS: Clinical evaluation of three families identified individuals with ASD in multiple generations. ASD was transmitted as an autosomal dominant trait in each family. ASD was the most common anomaly, but other heart defects occurred alone or in association with ASD in individuals from each kindred. Genome-wide linkage studies in one kindred localized a familial ASD disease gene to chromosome 5p (multipoint LOD score=3.6, theta=0.0). Assessment of 20 family members with the disease haplotype revealed that 9 had ASD, 8 were clinically unaffected, and 3 had other cardiac defects (aortic stenosis, atrial septal aneurysm, and persistent left superior vena cava). Familial ASD did not map to chromosome 5p in two other families. CONCLUSIONS: Familial ASD is a genetically heterogeneous disorder; one disease gene maps to chromosome 5p. Recognition of the heritable basis of familial ASD is complicated by low disease penetrance and variable expressivity. Identification of ASD or other congenital heart defects in more than one family member should prompt clinical evaluation of all relatives.

Rapid onset and dissipation of left atrial spontaneous echo contrast during percutaneous balloon mitral valvotomy.

BACKGROUND: Thromboembolism after percutaneous balloon mitral valvotomy (PBMV) has been attributed to dislodement of preexisting thrombus during transseptal puncture and instrumentation of the left atrium. The occurrence of thromboembolic events after PBMV in the absence of demonstrable left atrial thrombus before PBMV suggests that thrombus might form during the procedure. Spontaneous echo contrast (SEC) is a swirling pattern of blood echogenicity that is a marker of blood stasis in the left atrium. Exacerbation of left atrial SEC during PBMV may be indicative of an increased thromboembolic risk. METHODS: Transesophageal echocardiography was performed during PBMV in 20 patients with mitral stenosis. Grades of severity of left atrial SEC [0 (nil) to 4+ (severe)] were allocated before and after each balloon inflation. RESULTS: Before PBMV, SEC was present in 17 patients. New SEC or increased severity of SEC was observed during 49 of 56 balloon inflations. SEC was unchanged after six deflations, decreased after 14 deflations, and disappeared after 36 deflations. The mean times to onset and dissipation of SEC after balloon inflation and deflation were 3.1+/-1.5 and 3.9+/-1.6 seconds, respectively. After successful PBMV, SEC was unchanged in three patients, decreased in one, and resolved in 13. CONCLUSIONS: SEC is a dynamic and acutely reversible phenomenon that is highly sensitive to changes in left atrial hemodynamic conditions. Left atrial blood stasis induced by balloon inflation may promote thrombogenesis during PBMV.

Inhibition of red cell aggregation prevents spontaneous echocardiographic contrast formation in human blood.

BACKGROUND: Spontaneous echocardiographic contrast (SEC) is a pattern of blood echogenicity that has been attributed to ultrasonic backscatter from blood cell aggregates that form under low shear conditions. Patients with left atrial SEC have an increased thromboembolic risk. This study examined the role of red cell and platelet aggregates in the pathogenesis of SEC in human blood and the effects on SEC of antithrombotic therapy and red cell disaggregatory agents. METHODS AND RESULTS: Blood echogenicity was examined with the use of quantitative videodensitometry over a controlled range of flow velocities in an in vitro model characterized by nonlaminar flow conditions. One hundred ninety study samples were prepared from single fresh blood donations (40 to 120 mL) from 24 healthy volunteers and 11 patients. Whole blood echogenicity was unaltered by depletion of platelets, stimulation of platelet aggregation with adenosine diphosphate, or inhibition of platelet aggregation with aspirin. Low flow-related echogenicity increased with increasing hematocrit (P<.001) but was abolished when red cells were lysed selectively with saponin (P<.001). In the presence of red cells, low flow-related echogenicity increased with increasing fibrinogen concentration (P<.001) and with plasma paraproteins. Low flow-related echogenicity in whole blood was unaltered by heparin and warfarin but was reduced in a dose-dependent manner by dextran 40 (40 mg/mL, 70% reduction, P<.001) and poloxamer 188 (8 mg/mL, 47% reduction, P<.001), which inhibited red cell aggregation. CONCLUSIONS: These results support protein-mediated red cell aggregation as the mechanism of SEC in human blood. Inhibition of red cell aggregation, indexed by resolution of SEC, may provide an alternative to anticoagulant and antiplatelet therapy to reduce cardiac thromboembolic risk.

Direct effects of DC cardioversion on blood echogenicity: an in vitro study.

Exacerbation of left atrial spontaneous echo contrast (SEC) after cardioversion of atrial fibrillation has been attributed to left atrial mechanical dysfunction induced by the procedure ("atrial stunning"). An in vitro model was devised to determine whether electrically induced changes in blood properties might contribute to SEC formation after cardioversion. Human blood echogenicity was examined quantitatively by videodensitometry before and after shocks of 1, 2, 5, and 20 J. Changes in blood cell numbers, cell morphology, and erythrocyte sedimentation rate were determined by haematological analysis. Immediately following electrical discharges, transient and dose-related, highly echogenic microbubbles were noted, but shocks of increasing intensity did not induce SEC at high blood velocity or alter the severity of SEC at low blood velocity. No quantitative or qualitative changes in haematological parameters were observed. These results suggest that direct effects of electrical shock on blood do not contribute to SEC after cardioversion. Systemic haematological responses to electric shock that might indirectly promote red cell aggregation in vivo cannot be excluded by this in vitro study.