Using an in vitro motility assay system in an animal model, University of Tokyo investigators studied the role of myosin light chain in the motor function of cardiac myosin. The experiment involved the monitoring of the force induced by a single actin filament resulting from its interaction with myosin.

Seiryo Sugiura and colleagues initially compared the cardiac myosin isoforms V1 and V3, which are known to have distinct ATPase activity based on the heavy chain structure. They found that each force-producing event is shorter for the V1 isoform, which displays high ATPase activity. Next, using an in vitro expression system, the investigators studied the mutant myosins implicated in familial hypertrophic cardiomyopathy and found that the gene locus, which is close to the light chain interface, impacts heavily on motor function. This locus is relatively far from other functionally important domains such as the actin binding site.

Inspired by these results, Dr. Sugiura then studied the effect of atrial and ventricular light chains on the motor function of myosin in the rat model. The ATPase activity of these two isoforms as measured in solution was similar, but atrial myosin translocated the actin filaments faster and generated less force than ventricular myosin. The atrial myosin propelled the actin filament about 20% faster, Dr. Sugiura reported.

The experiment also showed that the longer the actin filament length, the more the myosin molecule participated in the generation of more force. According to the slope of this relationship, the ventricular isoform produced more force (higher time-averaged crossbridge force) than the atrial myosin. The question then became whether the greater force generated by ventricular myosin was due to a higher amplitude of each mechanical event or to its longer attachment time. Further experiments involving displacement under low and high load conditions showed that the distribution of force amplitude was similar between the two isoforms, however, the duration of each mechanical event was slightly longer for the ventricular myosin, as was the attachment time.

The mechanisms of this phenomenon remain unknown, however, it is possible that an essential light chain can form a link between thin and thick filaments to modulate the actomyosin interaction. The catalytic activity of myosin could be mainly determined by the heavy chain structure, but the myosin light chain also plays an important role as the modulator of kinetics, the investigators believe.

More than 18 genes have been associated with the occurrence of cardiomyopathy. The link between the genes and cardiomyopathic phenotypes is thought to be a calcium cycling defect. A Japanese/US multicenter study reported at this meeting explored possible mechanisms for this association.

The sarcoplasmic reticulum (SR) is integral to cardiac excitation-contraction coupling. There is re-uptake of calcium into the SR by SR calcium ATPase (SERCA2a), and decreased SERCA2a activity is a common feature of cardiomyopathy. To test the therapeutic value of enhancing calcium uptake, Susumu Minamisawa and colleagues generated several mutant phospholamban genes that disrupt the interaction between SERCA2a and phospholamban (phosphalamban is an endogenous inhibitor of SERCA2a). In genetically altered mice, phospholamban gene ablation prevented the development of cardiomyopathy functionally as well as morphologically. These results indicated that calcium cycling defects are critical in the progression of cardiomyopathy and that genetic defects can be linked to cardiomyopathic phenotypes, said Susumu Minamisawa, of the Heart Institute of Japan, Tokyo.

Looking for additional candidate genes, the investigators used direct sequencing analysis to explore mutations and single nucleotide polymorphisms of the SERCA2a and phospholamban genes in 99 cardiomyopathic patients. They found a single nucleotide transition, A to G at -77, of the phospholamban promoter region and two mutations of the SERCA2a gene in patients with hypertrophic cardiomyopathy. One mutation of the SERCA2a gene was a missense mutation in the cytoplasmic region, and the other was a single nucleotide transition, C to A, at the 5’ untranslated region. These mutations were not found in 131 control subjects, suggesting that SERCA2a and phospholamban are possible causal genes for hypertrophic cardiomyopathy.

The investigators then generated a pseudophosphorylated phospholamban mutant by replacing the serine 16 phosphorylation site with the basic amino acid glutamine, thereby introducing a negative charge at position 16 (S16E phospholamban mutant). The concept was verified when a transgenic serine 16/glutamate mutant mouse model displayed hypercontractility and relaxation when compared with control animals. Furthermore, in single cardiac cells the expression of the S16E phospholamban led to the improvement of contractility and relaxation in DCM-model mouse ventricular cells in the absence of catecholamines.

The investigators then tested whether the S16E phospholamban mutant improves in vivo cardiac function of the failing heart in a cardiomyopathic hamster model. The study used a new gene transfer technique that involved general hypothermia and intracoronary infusion of a recombinant adeno-associated viral vector. The percent fractional shortening of the left ventricle was better in the S16E phospholamban gene-transfected hearts, even at 7 months after the gene transfer.

Congestive heart failure is commonly caused by the disruption of the genome or physiome of dystrophin or sarcoglycan, a component in dystrophin-related proteins, according to a multicenter study presented by Tomie Kawada, of Niigata University Medical Hospital.

Dr. Kawada and colleagues reached this conclusion by studying three congestive heart failure models: (1) TO-2 strain hamsters with dilated cardiomyopathy undergoing gene therapy; (2) a rat model of acute congestive heart failure due to high-dose administration of isoproterenol; (3) a rat model of chronic heart failure secondary to previous myocardial infarction.

TO-2 strain hamsters with dilated cardiomyopathy demonstrate clinical symptoms identical to humans, in which the sarcoglycan gene is deleted. Sarcoglycan is a component of dystrophin-related proteins, and its deletion and corresponding protein dysfunction commonly induce muscle degeneration, as is the case in muscular dystrophy. The study used a recombinant adeno-associated virus (rAAV) vector that included normal sarcoglycan or Lac Z genes, driven by a cytomegalovirus promoter. The rAAV vectors were intramurally administered to the apex and left ventricular free wall of the 5-week-old hamsters during open-chest surgery. At 10 or 20 weeks, the animals underwent hemodynamic measurements, histological examination, immunostaining, and Northern and Western blottings and echocardiography at 30 weeks. The animals were followed for up to 40 weeks.

In vivo transfection of the rAAV vector with the normal sarcoglycan gene to the TO-2 hearts induced efficient expressions of both transgene and transcript in the myocardium. These results differed from Northern and Western blottings seen in control hamsters and in animals transfected only with the reporter gene. Hemodynamic indices by cardiac catheterization showed findings indicative of dilated cardiomyopathy in the TO-2 hamsters, but the delta-sarcoglycan transduction improved the parameters as compared to the group of animals transfected with the reporter gene only. The sarcoglycan transduction reduced the enlarged systolic dimension, and improved both the fractional shortening and ejection fraction accordingly.

Double fluorescence microscopy identified the myocardial cells presenting delta-sarcoglycan or taking up exogenously applied Evans blue dye. Cardiac muscle transduced by the sarcoglycan gene revealed the expression of the transgene, but dye was not detected in this sample, indicating the preservation of the sarcolemmal integrity. The sarcoglycan gene treatment of the TO-2 strain hamsters protected the cardiomyocytes from sarcolemmal leakage in situ.

The novel gene treatment also improved survival by Kaplan Meier analysis, as five animals in the reporter gene group died by 35 weeks while all animals in the delta-sarcoglycan gene replacement group survived and remained active. The authors concluded that the gene therapy prolonged survival because the responsible gene causing the dilated cardiomyopathy was supplemented in vivo. These results suggest that the genome mutation modifies the proteome expression and finally alters the corresponding physiome, and that the deterioration of the physiome could be halted by the transduction of a new genome.

In additional studies, isoproterenol caused disruption of dystrophin but not delta-sarcoglycan. Dystrophin was translocated to myoplasm and fragmented, while delta-sarcoglycan was preserved in sarcolemma and was not hydrolyzed. Dystrophin, alpha-sarcoglycan and gamma-sarcoglycan, but not delta-sarcoglycan, were reduced in survived myocardium after coronary ligation. ACE inhibitors ameliorated the decrease of dystrophin and alpha-sarcoglycan but not gamma-sarcoglycan in vivo. Isolated m-calpain selectively degraded dystrophin, alpha-sarcoglycan, and gamma-sarcoglycan but not delta-sarcoglycan.

These findings all suggest that congestive heart failure is commonly caused by disruption of the genome or physiome of dystrophin or sarcoglycan, irrespective of hereditary or acquired origins. Somatic gene therapy combined with pharmacotherapy is a promising approach to preventing advanced disease.