Temporal alternations in force-dependent and velocity-dependent indices of cardiac contractility in the evolution of diabetic cardiomyopathy: roles of oxidative stress, nitrosative stress, myosin heavy chain isoform switch and oxidative-sensitive transcri

• Main Authors: Dong-Feng Yeih, 葉東峰
• Other Authors: 曾淵如
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/47515528569624025569

博士 === 臺灣大學 === 生理學研究所 === 98 === Diabetes mellitus may result in impaired cardiac contractility, but the underlying mechanical and molecular mechanisms remain unclear. There are three major aims of the present doctoral thesis. First, we aimed to investigate the temporal alternations in cardiac mechanics (i.e., force-dependent and velocity-dependent indices of cardiac contractility) in the evolution of diabetic cardiomyopathy (DCM) in terms of the elastance-resistance left ventricle (LV) pump model. In addition, we investigated the temporal changes in loading conditions, mechanical efficiency and ventriculoarterial coupling to elucidate the detailed cardiac mechanics attributable to decompensated heart failure in streptozotocin (STZ)-induced diabetic rats. Second, we aimed to evaluate the preventive and therapeutic effects of dimethylthiourea (DMTU), a potent hydroxyl radical scavenger, on force-dependent and velocity-dependent indices of cardiac contractility in both early and chronic stages of STZ-diabetic rats. We sought to do this in terms of force-dependent and velocity-dependent indices of myocardial contractility by using the elastance-resistance LV model. Furthermore, we planned to carry out experiments to examine downstream transcription factors, such as myocyte enhancer factor-2 (MEF-2) and heart autonomic nervous system and neural crest derivatives (dHAND and eHAND), activated by oxidative stress and markers of oxidative stress and the expression of isoforms of myosin heavy chain (MHC) in STZ-diabetic rat hearts. Third, we also evaluated the therapeutic efficacy of NG-nitro-L-arginine methyl ester (L-NAME), a non-specific inhibitor of nitric oxide synthases (NOS), in STZ-diabetic rats in terms of the elastance and resistance of left ventricle (LV). Furthermore, we planned to carry out experiments to investigate the underlying molecular mechanisms by examining the levels of nitrosative stress and oxidative stress markers, and its downstream signaling, i.e., nuclear factor-κB (NFκB), and coupling of NOS, as well as modulation of MHC isoform in STZ-diabetic rats. In the first part, we aimed to investigate the temporal alterations in cardiac mechanics, loading conditions as well as mechanical efficiency in the evolution of systolic dysfunction in STZ-diabetic rats. Adult male Wistar rats were randomized into control and STZ-induced diabetic groups. Invasive hemodynamic studies were done at 8, 16, and 22 weeks post STZ injection. Maximal systolic elastance (Emax) and maximum theoretical flow (Qmax) were assessed by curve fitting techniques; ventriculoarterial coupling and mechanical efficiency by a single beat estimation technique. In contrast to early occurring and persistently depressed Emax, Qmax progressively increased with time, but was decreased at 22 wks post STZ injection, which temporally correlated with the changes in cardiac output. The favorable loading conditions enhanced stroke volume and Qmax, while ventriculoarterial uncoupling attenuated the cardiac mechanical efficiency in diabetic animals. The changes in Emax and Qmax are discordant during the progression of contractile dysfunction in the diabetic heart. Our present study showed that attenuated afterload-adjusted Qmax (Qmaxad) and afterload-adjusted LV weight normalized Qmax (Qmaxadn) and cardiac mechanical efficiency, occurring preceding overt systolic heart failure, are two major determinants of deteriorating cardiac performance in diabetic rats. After validation of the usefulness of both force-dependent and velocity-dependent indices of cardiac contractility in diabetic cardiomyopathy, we then aimed to investigate the preventive and therapeutic effects of DMTU on cardiac mechanics in STZ-diabetic rat hearts. It has been reported that hydroxyl radicals and hydrogen peroxide are involved in the pathogenesis of systolic dysfunction in diabetic rats, but the precise mechanisms and the effect of antioxidant therapy in diabetic subjects have not been elucidated. We aimed to evaluate the effects of DMTU on both force-dependent and velocity-dependent indices of cardiac contractility in STZ-induced early and chronic diabetic rats. Seventy-two hours and eight weeks after STZ (60 mg/kg) injection, diabetic rats were randomized to either DMTU (50 mg/kg/day, IP) or vehicle treatment for 6 and 12 weeks, respectively. All rats were then subjected to invasive hemodynamic studies. Again, Emax and Qmax were assessed by curve fitting techniques in terms of the elastance-resistance model. Both LV weight normalized Emax (Emaxn) and Qmaxad were depressed in diabetic rats, concomitant with altered MHC isoform composition and its upstream regulators, such as MEF-2 and heart autonomic nervous system and neural crest derivatives (eHAND and dHAND). In chronic diabetic rats, DMTU markedly attenuated the impairment in Qmaxad, and normalized the expression of MEF-2 and eHAND, and MHC isoform composition, but exerted an insignificant benefit on Emaxn. Regarding preventive treatment, DMTU significantly ameliorated both Emaxn and Qmaxad in early diabetic rats. We also checked blood glucose and insulin concentrations in controls and diabetic rats. The results showed that plasma insulin levels were significantly decreased after STZ injection and blood glucose levels were significantly increased in diabetic rats, but there were no significant differences after DMTU treatment in diabetic groups. In the second part, our current study shows that the advantage of DMTU in chronic diabetic rats might involve normalization of MEF-2 and eHAND, as well as reversal of MHC isoform switch. Finally, we evaluated the therapeutic efficacy of L-NAME, a non-specific inhibitor of NOS, in STZ-diabetic rats in terms of the elastance and resistance of left ventricle. Several reports have shown that nitrosative stress (NS) plays an essential role in diabetic cardiomyopathy. However, the precise mechanism by which the NS leads to compromised cardiac contractility has not been elucidated. We aimed to test the hypothesis that uncoupled endothelial NOS (eNOS) under excessive nitrosative stress and oxidative stress may enhance the translocation of NFκB and the resultant MHC proteolysis and isoform switch. Four weeks after STZ (60 mg/kg) or vehicle injection, male Wistar rats were randomized to receive treatment with either L-NAME (30 mg/kg/day in drinking water) or vehicle for another 8 weeks and then followed by invasive hemodynamic studies. Similarly, both Emax and Qmax were assessed by curve fitting techniques; the Ea by a single beat estimation technique. Analysis of low temperature sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE) of eNOS and Western blotting of NFκB-p65 in nuclear extract of the LV were done in the control, STZ and STZ+L-NAME groups. In parallel, catalase and oxidized to reduced glutathione ratio (GSSG/GSH ratio), and 3-nitrotyrosine (3-NT) in the LV were also measured. Both Emaxn and Qmaxadn were significantly depressed in 12-week diabetic rats, accompanied with nuclear translocation of NFκB-p65 and MHC isoform switch. L-NAME treatment not only attenuated the reductions in both Emaxn and Qmaxadn in diabetic rats, but also significantly modulated NFκB translocation, and MHC isoform switch analyzed by real time polymerase chain reaction (RT-PCR). We demonstrated that chronic inhibition of NOS reduced uncoupled eNOS and levels of NS and oxidative stress markers on one hand, and improved both in vivo force-dependent and velocity-dependent indices of cardiac contractility on the other hand. Our results suggested that NS might play an essential role in the pathogenesis of diabetic cardiomyopathy by mediating the activation of NFκB, and subsequent MHC isoform switch. In conclusion, the present doctoral thesis combined cardiac mechanics studies and molecular studies to demonstrate the possible mechanisms attributable to overt heart failure in the evolution of diabetic cardiomyopathy. First, at early stage of DCM, the enhanced Qmax and favorable loading conditions play complementary roles to persistently depressed Emax, and cardiac performance is then well-preserved in early period of diabetic rats. However, Qmax is attenuated at later stages, which is temporally correlated with the declines in stroke volume and cardiac output. The compensatory offset between Emax and Qmax would be lost and depressed cardiac performance would then ensue. Like systolic elastance, Qmax can serve as a velocity-dependent dimension of cardiac contractile function and can predict the occurrence of overt systolic dysfunction. In addition, cardiac mechanical efficiency, rather than aortic hydraulic energy transfer, is diminished preceding overt systolic heart failure, and may play a detrimental role in the evolution of contractile dysfunction in STZ-induced diabetic rats. By unraveling the temporal changes in Emax and Qmax, and loading conditions as well as cardiac mechanical efficiency, our present study provides a comprehensive understanding of the pathogenesis of contractile dysfunction in diabetic rat heart. It has been reported that isoform switch of MHC might play a role in depressed Qmaxad, it warrants further study to elucidate the possible molecular mechanisms attributable to attenuated Qmaxad, such as extracellular component (i.e., collagen), and geometric factors (i.e., concentric ventricular hypertrophy). Second, we demonstrated that DMTU normalizes Emaxn and Qmaxad in early stage of DCM, but normalizes only Qmaxad in late stage of diabetic cardiomyopathy. DMTU, by reducing lipid peroxidation and enhancing antioxidant capacity, has disparate effects on modulation of the myocardial response to increased oxidative stress in chronic diabetic rat hearts, with a dramatic restoration of velocity-dependent index of cardiac contractility but an insignificant benefit on force-dependent index. The advantages of DMTU treatment might involve normalization of cardiac-specific transcription factors, such as MEF-2 and eHAND, as well as the reversal of MHC isoform switch in chronic diabetic rats. These observations suggest a way towards an additional therapeutic approach to systolic dysfunction in diabetes. Finally, we showed that chronic inhibition of NOS may ameliorate both force-dependent and velocity-dependent indices of cardiac performance in diabetic rats. The underlying molecular mechanisms of L-NAME treatment might be mediated through attenuation of uncoupled eNOS and nitrosative/oxidative stress, and NFκB translocation from the cytosol to the nucleus, and subsequent reversal of MHC isoform switch.