A. Malhotra

Effect of aging and hypertension on myosin biochemistry and gene expression in the rat heart.

The mechanisms by which the aged heart adapts to a superimposed pressure load such as hypertension have not been described. We therefore investigated biochemical and molecular genetic adaptations in the 24-month-old rat heart subjected to renovascular hypertension. Compared with 4-month-old rats, aging was associated with a 68% increase in left ventricular mass without any change in heart weight-to-body weight ratio, a 33% reduction in calcium-activated myosin ATPase activity, and a shift from a V1 to a V3 predominant myosin heavy chain (MHC) isoform distribution. A 46% reduction in alpha-MHC mRNA and a reciprocal increase in beta-MHC mRNA was seen. When hypertension was superimposed, there was a further 75% increase in ventricular mass, a 63% increase in heart weight-to-body weight ratio, and a 19% reduction in myosin ATPase. Myosin isozyme distribution was further shifted to V3, and the ratio of alpha-MHC to beta-MHC mRNA was reduced. In addition, with hypertension a significant (greater than 50%) reduction in the mRNA level of the cardiac sarcoplasmic reticular calcium-activated ATPase was seen. These data demonstrate that the aged myocardium is able to respond to a superimposed pressure load with a molecular genetic and protein synthetic pattern of hypertrophy analogous to that seen in younger animals.

Effects of chronic dobutamine administration on hearts of normal and hypertensive rats.

We have previously shown that physical conditioning in the rat improves cardiac mechanics and biochemistry and normalizes the cardiac contractile protein abnormalities associated with renovascular hypertension. Since chronic adrenergic stimulation with dobutamine simulates some aspects of physical conditioning, this study was undertaken to investigate the effects of chronic dobutamine administration on normal and hypertensive rat hearts. Four groups of female animals were studied: controls, dobutamine-treated (2 mg/kg twice daily), renovascular hypertensives, and dobutamine-treated hypertensives. Animals were killed after 8-10 weeks and cardiac histology, myosin biochemistry, and mechanics in an isolated heart perfusion apparatus were studied. Dobutamine, unlike hypertension, was not associated with histological evidence of myocardial damage but did increase cardiac mass by 10% and calcium-activated myosin ATPase activity by 13%. Hypertension was associated with a 24% increase in mass, a 24% decrease in ATPase activity, and a shift in the myosin isoenzyme pattern from V1 to V3. The combined stimuli caused additive hypertrophy (44%) and normalized myosin biochemistry and isomyosin distribution. Dobutamine treatment was not associated with significant improvements in pump or muscle function in control or hypertensive hearts. Thus chronic dobutamine treatment, like physical conditioning, induces a physiological cardiac hypertrophy in rats that is associated with improved myosin enzymology and normalization of the contractile protein abnormalities associated with hypertension. Unlike physical conditioning, however, these biochemical alterations do not result in improved contractile function as measured in an isolated buffer-perfused heart apparatus.

Troponin subunits contribute to altered myosin ATPase activity in diabetic cardiomyopathy

Our group has documented that myocardial performance is impaired in the hearts of chronically diabetic rats and rabbits. Abnormalities in the contractile proteins and regulatory proteins may be responsible for the mechanical defects in the streptozotocin (STZ)-diabetic hearts. Previously, the major focus of our research on contractile proteins in abnormal states has concentrated on myosin ATPase and its isoenzymes. Our present study is based on the overall hypothesis that regulatory proteins, in addition to contractile protein, myosin contribute to altered cardiac contractile performance in the rat model of diabetic cardiomyopathy. The purpose of our research was to define the role of cardiac regulatory proteins (troponin-tropomyosin) in the regulation of actomyosin system in diabetic cardiomyopathy.For baseline data, myofibrillar ATPase studies were conducted in the myofibrils from control and diabetic rats. To focus on the regulatory proteins (troponin and tropomyosin), individual proteins of the cardiac system were reconstituted under controlled conditions. By this approach, myosin plus actin and troponin-tropomyosin from the normal and diabetic animals could be studied enzymatically. The proteins were isolated from the cardiac muscle of control and STZ-diabetic (4 weeks) rats. Sodium dodecyl sulfate gel electrophoretic patterns demonstrate differences in the cardiac TnT and TnI regions of diabetic animals suggesting the different amounts of TnT and/or TnI or possibly different cardiac isozymes in the regulatory protein complex. Myofibrils probed with a monoclonal antibody TnI-1 (specific for adult cardiac TnI) show a downregulation of cardiac TnI in diabetics when compared to its controls. Enzymatic data confirm a diminished calcium sensitivity in the regulation of the cardiac actomyosin system when regulatory protein(s) complex was recombined from diabetic hearts. Actomyosin ATPase activity in the hearts of diabetic animals was partially reversed when myosin from diabetic rats was regulated with the regulatory protein complex isolated from control hearts. To our knowledge, this is the first study which demonstrates that the regulatory proteins from normal hearts can upregulate cardiac myosin isolated from a pathologic rat model of diabetes. This diminished calcium sensitivity along with shifts in cardiac myosin heavy chain (V1→V3) may be partially responsible for the impaired cardiac function in the hearts of chronic diabetic rats. (Mol Cell Biochem151: 165–172, 1995)

Increased heart rate prevents the isomyosin shift after cardiac transplantation in the rat.

The heterotopically transplanted rat heart undergoes significant atrophy and a shift from V1 to V3 isomyosin. The purpose of this study was to pace the cardiac isograft and determine whether an increase in heart rate would attenuate the changes in cardiac mass and isoenzyme distribution. Nonpaced transplanted hearts were compared with hearts in which pacing was initiated at 7 Hz, 24 hours after transplantation, and continued for 7 days. There was a 29% decrease in myosin ATPase activity and a 22% decrease in alpha-myosin in the nonpaced isograft; both decreases were completely prevented by pacing. The decrease in cardiac mass was also significantly attenuated. Pacing did not alter intrinsic heart rate, systolic pressure, dP/dt, or norepinephrine concentration in the isograft. These results suggest that the adaptation in both cardiac mass and isoenzymes may be related to the rate or the rate-pressure product in the transplanted paced heart independent of left ventricular pressure, tissue catecholamines, or neural activity.

ROLE OF REGULATORY PROTEINS (TROPONIN-TROPOMYOSIN) IN PATHOLOGIC STATES

In vertebrate striated muscle, troponon-tropomyosin is responsible, in part, not only for transducing the effect of calcium on contractile protein activation, but also for inhibiting actin and myosin interaction when calcium is absent. The regulatory troponin (Tn) complex displays several molecular and calcium binding variations in cardiac muscles of different species and undergoes genetic changes with development and in various pathologic states.Extensive reviews on the role of tropomyosin (Tm) and Tn in the regulation of striated muscle contraction have been published describing the molecular mechanisms involved in contractile protein regulation. In our studies, we have found an increase in Mg2+ ATPase activity in cardiac myofibrils from dystrophic hamsters and in rats with chronic coronary artery narrowing. The abnormalities in myofibrillar ATPase activity from cardiomyopathic hamsters were largely corrected by recombining the preparations with a TnTm, complex isolated from normal hamsters indicating that the TnTm, may play a major role in altered myocardial function. We have also observed down regulation of Ca2+ Mg2+ ATPase of myofibrils from hypertrophic guinea pig hearts, myocardial infarcted rats and diabetic-hypertensive rat hearts. In myosin from diabetic rats, this abnormality was substantially corrected by adding troponin-tropomyosin complex from control hearts. All of these disease models are associated with decreased ATPase activities of pure myosin and in the case of rat and hamster models, shifts of myosin, heavy chain from alpha to beta predominate.In summary, there are three main troponin subunit components which might alter myofibrillar function however, very few direct links of molecular alterations in the regulatory proteins to physiologic and pathologic function have been demonstrated so far.

Swimming causes myosin adaptations in the rat cardiac isograft.

To investigate the contributions of humoral and hemodynamic factors to cardiac adaptations associated with chronic exercise, female Fischer 344 rats were subjected to chronic swimming, infrarenal cardiac transplantation, or both. Swimming resulted in hypertrophy (11-12%) of the in situ hearts in both the unoperated and operated animals compared with the matched sedentary controls. The cardiac isograft exhibited atrophy (32-35%), which was not attenuated by swimming. The cardiac isograft was also associated with a decrease in the percent of V1 myosin isoenzyme, which was attenuated by swimming (45 +/- 5% versus 66 +/- 6%). Swimming also increased the percent of this isomyosin in the in situ hearts of operated rats. These data suggest that hemodynamic load and/or neural innervation are necessary for hypertrophy associated with chronic conditioning by swimming, whereas myosin isoenzyme control is significantly mediated by humoral factors.