The Related Molecular Mechanisms About The Transition Of Pathological Cardiac Hypertrophy To Heart Failure

Pathological cardiomyocyte hypertrophy is the common response when the heart endures damage factors. Persistent hypertrophy eventually leads to cardiac remodeling, chamber dilation and cardiac dysfunction. There is a growing interest in identifying the signaling mechanisms underlying the development of cardiac hypertrophy and the transition to heart failure.Numerous studies have suggested a role of ROCK in mediating cardiac hypertrophy and remodeling by using pharmacological inhibitors of ROCK. The first chapter of this thesis will focus on the potential role of ROCK1, one of the two isoforms of ROCK family, on the cardiac remodeling in pathological hypertrophy model via in vivo loss-of-function of ROCK1.Cardiomyocyte apoptosis has been considered as an essential factor during the transition of cardiac hypertrophy to heart failure. However, even under pathological conditions, the absolute prevalence of cardiomyocyte apoptosis is still low. Thus, the significance of apoptosis to human ventricular remodeling continues to be debated. Based on this consideration, in the second chapter we will investigate the significance of cardiomyocyte apoptosis in cardiomyopathy at different ages, through elucidating the age-related difference of cardiomyocyte apoptosis under physiological and pathological conditions.Chapter Ⅰ Disruption of ROCK1gene attenuates cardiac dilation and improves cardiac function in pathological cardiac hypertrophyBACKGROUNDPathological cardiomyocyte hypertrophy is the common response when the heart endures increased hemodynamic load, myocardial injury, and neurohormonal stress. Persistent stress eventually leads to a decompensate congestive heart failure, which is characterized by chamber dilation and myocardial dysfunction. There is a growing interest in identifying the signaling mechanisms underlying the development of cardiac hypertrophy and the transition to heart failure.The Gq class of heterotrimeric G proteins is one important signal transducer that is responsible for the development of cardiac hypertrophy and subsequent cardiac decompensation. Transgenic overexpression of the α subunit of Gq in the myocardium is a well-characterized in vivo pathological hypertrophy model which recapitulates many cellular, molecular, and functional characteristics of pressure overload induced hypertrophy including left ventricular dilation, depressed ventricular contractility at baseline and in response to β-adrenergic receptor stimulation, diminished basal and agonist stimulated adenylyl cyclase (AC) activity, and activation of a fetal cardiac gene program. A number of studies have delineated several potential mechanisms by which Gocq overexpression leads to cardiomyocyte hypertrophy, cardiac dilation, and dysfunction. These mechanisms include increased expression and activity of protein kinase C (PKC)α, decreased AC expression and catalytic activity, and increased activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) kinase kinase1(MEKK1)/c-Jun N-terminal kinase (JNK) signaling pathway.Rho-associated coiled-coil containing protein kinase (ROCK) is a downstream mediator of RhoA GTPase, and is believed to play a critical role in mediating the effects of RhoA on stress fiber formation, smooth muscle contraction, cell adhesion, membrane ruffling, and cell motility. Recent in vivo studies using ROCK inhibitors, Y27632and fasudil, suggest a role of ROCK in mediating cardiac hypertrophy and remodeling. However these pharmacological inhibitors are not able to distinguish between the two isoforms of the family, ROCK1and ROCK2. We have recently shown that ROCK1homozygous deficient mice developed cardiac myocyte hypertrophy in response to pressure overload induced by transverse aortic constriction, indicating that ROCK1is not critical for the development of cardiac hypertrophy. However these mice exhibited significantly reduced interstitial fibrosis and cardiomyocyte apoptosis. This observation is consistent with another recent study using ROCK1haploinsufficient mice which did not show decreased hypertrophy but decreased perivascular fibrosis induced by angiotensin Ⅱ. A number of the previous in vitro studies performed in cultured rat neonatal cardiomyocytes have shown that RhoA/ROCK pathway cooperated with Gαq pathway for the induction of cardiomyocyte hypertrophy by G protein-coupled receptor agonists. The combination of the well-characterized transgenic Gαq cardiac hypertrophy model with the ROCK1deficient mice provides an opportunity to examine the potential in vivo interaction between ROCK1and Gαq pathways and to test effects of ROCK1deletion on the development of dilated cardiomyopathy induced by intrinsic cardiac effects in Gαq overexpression transgenic mice.Part One ROCK1deletion prevents cardiac dilation and improves contractile function in Gαq miceMETHODS(1) Survival study:Sex (male)-matched FVB mice were divided into4groups (n=5): wild type group (WT), cardiac specific Gαq overexpressing group (Gαq), Gαq-oevrexpressing and ROCK1-knockout compound group (Gαq/ROCK1-/-), and ROCK1-knockout group (ROCK1-/-). All these mice underwent echocardiography at the age of10months, and were followed for12months.(2) Other protein analyses, histological and functional analyses:Male FVB mice were divided into4groups:wild type (WT)、Gαq、 Gαq/ROCK1-/-and ROCK1-/-. All these mice underwent echo-cardiography and were sacrificed at the age of12weeks, and the hearts were saved for western blot analysis and histological analysis.RESULTS1. Expression levels of Gαq and ROCK:(1)Gαq expression level was increased significantly and invariant in Gαq and Gαq/ROCK1-/-mice. ROCK1deficiency did not change the expression level of Gαq transgene in ventricular tissue.(2)ROCK1expression was absent in ROCK1-/-and Gαq/ROCK1-/-mice.(3)ROCK2expression remained similar in all four groups.(4)No significant difference was detected for ROCK activity assessed by Western blot analysis among these groups.2. ROCK1deletion prolonged the survival time of Gαq mice significantly. 3. Disruption of ROCK1did not prevent development of cardiomyocyte hypertrophy induced by cardiac-specific overexpression of Gαq.4. ROCK1deletion partially prevented the induction of embryonic marker genes by cardiac-specific overexpression of Gαq.5. ROCK1deletion prevented left ventricular dilation and dysfunction induced by cardiac-specific overexpression of Gαq.CONCLUSIONROCK1deficiency prevents cardiac dilation, improves cardiac function, and prolongs survival time of cardiac-specific Gαq overex-pression transgenic mice. METHODS4groups of FVB mice were enrolled in this study:WT、Gαq、Gαq/ROCK1-/-and ROCK1-/-. All these mice were sacrificed right after undergoing echocardiography under baseline or Isoproteronol stimulation at the age of12weeks. Ventricular tissue homogenate was used for western blot analysis, and whole heart was used for histological analysis.RESULTS1. Fibrosis was not consistently observed in the Gαq and Gαq/ROCK1-/cardiac tissue.2. There was no significant increase in cardiac apoptosis in Gαq and Gαq/ROCK1-/-groups compared with WT or ROCK1-/-groups.3. Gαq/ROCK1-/-mice exhibited improved contractility in response to β-adrenergic receptor stimulation when compared with Gαq mice.4. ROCK1deletion did not inhibit MAPK activation and PKCα activation induced by cardiac-specific overexpression of Gαq.5. ROCK1deletion rescued down-regulation of adenylyl cyclase type Ⅴ/Ⅵ induced by cardiac-specific overexpression of Gαq.CONCLUSIONROCK1deletion prevents down-regulation of adenylyl cyclase type Ⅴ/Ⅵ and β-adrenergic dysfunction induced by cardiac-specific overexpression of Gαq, which is consistent with improved contractile function by ROCK1deletion. CONCLUSIONS OF CHAPTER ⅠROCK1deletion prevents cardiac dilation, down-regulation of adenylyl cyclase type Ⅴ/Ⅵ and β-adrenergic dysfunction induced by cardiac-specific overexpression of Gαq, which is consistent with the improved contractile function and prolonged survival time by ROCK1deletion.Chapter Ⅱ The age-related difference of cardiomyocyte apoptosis and the relationship between cardiomyocyte apoptosis and cardiomyopathy at different agesPart One The age-related difference of caspases-dependent cardiomyocyte apoptosisBACKGROUNDTwo canonical pathways involved in caspases-dependent apoptosis include the extrinsic pathway and the intrinsic pathway, which are initiated through activation of caspase8and caspse9respectively, and converge on the cleavage of apoptotic executioner caspases3,6,7. In the intric pathway, Apaf1serves as adaptor protein to link cytochrome C and pro-caspase9, and is necessary to the activation of pro-caspase9. Bcl-2family, which is believed to be a major regulator, contains pro-apoptotic proteins, such as Bax, Bak, BH3-only proteins, and anti-apoptotic proteins, such as Bcl-2, Bcl-XL and Mcl-1.Cardiomyocyte apoptosis is essential to the embryonic cardiac development, and becomes very rare after birth. Numerous studies have demonstrated the significance of cardiomyocyte apoptosis in cardiomyopathy, however even under the pathological conditions, the absolute prevalence of cardiomyocyte apoptosis is still very low. Another question which has been attracting much attention is that the young hearts present more susceptibility to the cardiac damages than the adult hearts. We speculate that the age-dependent changes of the cell death and proliferation-related genes and proteins may be linked to the difference of the sensitivity to stimuli between young and adult hearts.At first, the changes of the cardiomyocyte apoptosis and the apoptotic molecules, including caspase3, caspase8, caspase9, Apaf1, Bax, etc, were determined from the neonate to the adult under physiological conditions; Furthermore, the combination of the specific labeling of cardiomyocyte nuclei in which the lacZ gene is overexpressed and in situ TUNEL staining gave us an access to precisely quantify the cardiomyocyte apoptosis at different ages.METHODSFVB wild type mice (WT) and transgenic FVB mice overexpressing lacZ in the nuclei of cardiomyocytes (αMHC-nlacZ) were enrolled in this study. About the caspase3activity measurement and the caspases protein analysis,6groups of WT mice were included:1-day-old group,3-day-old group,1-week-old group,2-week-old group,3-week-old group and5-week-old group. Regarding the immunostaining of active-caspase3, TUNEL staining and other protein analysis,3groups of WT or nlacZ mice were included:1-week-old group,3-week-old group and5week-old group. The β-gal staining to specifically label the cardiomyocytes was performed before immuno staining and TUNEL staining.RESULTS1. The nuclei of cardiomyocytes in αMHC-nlacZ presented blue color after β-Gal staining compared with the cytosol and other types of cells.2. Caspase3activity assay showed that the caspase3activity in myocardium was decreased significantly with the age. The caspase3 activity was much higher in1-week-old cardiac tissue, decreasing and going to be undetectable after3weeks.3. Immunostaining assay and western blot showed that the expression level of active caspase3in cardiomyocytes was decreased significantly with the age, and no active caspase3was detected in the cardiomyocytes after3weeks.4. Combination of β-Gal staining and TUNEL staining showed that the cardiomyocyte apoptosis was decreased significantly with the age. Some apoptotic cardiomyocytes could be observed at the age of1week, however rare cardiomyocyte apoptosis occurred3weeks after birth.5. From the results of western blot, the expression level of pro-caspase3was much higher in1-week-old cardiac tissue, and decreased significantly after2weeks, and became very weak at the age of3weeks; also, the expression levels of pro-caspase9and pro-caspase8were decreased significantly with the age; Active-caspase9, but no active-caspase8, was detected in1-week-old myocardium, but not in3-and5-week-old myocardium.6. From the results of western blot, the expression levels of other proteins which are essential for the intrinsic apoptotic pathway, including Bax and Apafl, were significantly down-regulated with the age. 7. The expression of other proliferation-and survival-related proteins, such as Akt, p-Akt, p-P53, p-p38and p-p42/44, did not present the age-dependent change.CONCLUSIONIn the earlier stage of the postnatal cardiac development, the cardiac apoptotic potential is much higher than that in the adult hearts, which may render a greater tendency towards cardiac apoptosis in the neonatal heart with damage, and may serve as one of the pivotal mechanisms for the higher susceptibility of the neonatal heart to the cardiac damage. BACKGROUNDDoxorubicin (DOX) is one of the most widely used and successful antitumor drugs. Cardiotoxicity is a major limiting factor in anticancer therapy. Numerous studies indicate that cardiomyocyte apoptosis plays a key role in the DOX-induced cardiotoxicity. However, most of the in vivo studies about DOX-induced cardiotoxicity only focus on the adult hearts, in which the rate of cardiomyocyte apoptosis is very low, even under the pathological conditions. In addition, all the methods to measure the cardiomyocyte apoptosis in situ are unable to precisely distinguish cardiomyocytes and non-cardiomyocytes. Thus, the significance of apoptosis to cardiomyopathy and ventricular remodelling induced by DOX or other stimuli continues to be debated.As demonstrated by clinical studies, children and adolescents are particularly susceptible to the cardiotoxic effects of DOX. The possible rationale was due to the loss of myocytes and impaired cardiac growth resulting in inadequate left ventricular mass and cardiomyopathy a year or more after cessation of chemotherapy. The results from the preceding section and other studies suggest that the more active intrinsic apoptotic pathway, particularly the higher expression level of pro-caspase3, may contribute to the more severe DOX cardiotoxicity in the neonatal hearts.In the present study, we further observed the difference of the cardiomyocyte apoptosis and the apoptotic pathways between neonatal and adult hearts at the earlier stage of DOX-induced cardiotoxicity, precisely quantified the DOX-induced cardiomyocyte apoptosis via combining the β-gal staining and TUNEL staining, and measured the significance of cardiomyocyte apoptosis to cardiomyopathy at different ages. METHODSFVB-WT and aMHC-nlacZ mice were recruited in this study.(1)The effects of DOX with different concentrations on the expression of caspase3in1-week-old mouse hearts: Mice were divided into5groups (n=4):Saline-treated group (1wk-ctrl), DOX5mg/kg-treated group (1wk+5), DOX10mg/kg-treated group (1wk+10), DOX15mg/kg-treated group (1wk+15), DOX20mg/kg-treated group (1wk+20). DOX was given by i.p injection. Mice were sacrificed to harvest hearts24h after injection.(2) The time-course analysis of effects of DOX10mg/kg on the caspase3expression in1-week-old hearts: Mice were divided into7groups:treated with DOX for0hour,2,4,8,12,16,24hours (n=4).(3) The effects of DOX with low (10mg/kg) and high (20mg/kg) concentrations on cardiomyocyte apoptosis and apoptotic pathways in mouse hearts with different ages: Mice were divided into9groups:1wk-ctrl,1wk+10,1wk+20;3wk-ctrl,3wk+10,3wk+20;5wk-ctrl,5wk+10,5wk+20.RESULTS1. The expression levels of active caspase3and the production of cleaved PARP were increased in a DOX concentration-dependent manner in the1-week-old mouse hearts, and became significantly higher with the dose increasing to10mg/kg.2. The expression levels of active caspase3were induced gradually by DOX, and peaked at24hours after DOX administration in1-week-old myocardium.3. The caspase3activity was increased significantly in the1-week-old myocardium after treatment with low-dose DOX (10mg/kg), and further higher in the high-dose group (20mg/kg).4. The rate of cardiomyocyte apoptosis was increased significantly in the1-week-old mouse hearts in a DOX concentration-dependent manner.5. The expression level of active caspase9was increased significantly in a concentration-dependent manner after treatment with DOX in1-week-old hearts.6. The expression of Bax was induced significantly by DOX treatment in both neonatal and adult hearts.7. The caspase3activity was undetectable in3-and5-week-old hearts treated with DOX10or20mg/kg for24hours.8. The expression of active caspase3was undetectable in3-and5-week-old hearts after treatment with DOX10or20mg/kg for24hours.9. The cardiomyocyte apoptosis was not present in3-and5-week-old hearts treated with DOX10or20mg/kg for24hours.10.The expression of active caspase9was undetectable in3-and5-week-old hearts treated with DOX10or20mg/kg for24hours.11.DOX exerted no effects on the expression of pro-caspase3, pro-caspase9, Apaf1, XIAP, pro-caspase8and active-caspase8in both neonata1and adult hearts.12.DOX exerted no effects on the expression of Akt/p-Akt, p-P53, p-p38, p-p42/44in both neonatal and adult hearts.CONCLUSIONThe higher activity of apoptotic pathways leads to a higher occurrence of apoptosis in the neonatal heart after DOX treatment, suggesting that excessive cardiomyocyte apoptosis is one of the primary and major factors involved in the earlier cardiotoxicity induced by DOX in the juvenile hearts, and is one of the underlying mechanisms by which the neonatal hearts are exposed to a high susceptibility to the DOX-induced cardiotoxicity. Our present in vivo study suggests for the first time that the cardiac apoptotic potential is much higher in the neonatal hearts than that in the adult hearts, which is one of the primary and major factors involved in the earlier cardiotoxicity induced by DOX in the juvenile hearts, and is one of the underlying mechanisms by which the juvenile patients are exposed to a higher susceptibility to the DOX-mediated cardiotoxicity.