Experimental Proof For "Myocardial Hypertrophic Preconditioning"

Background and Objective:Characterized with increased cardiomyocyte protein synthesis and increased cell volume, myocardial hypertrophy is crucial for the transition of cardiac function from adaptive to maladaptive status and progression to irreversible change. In a broad sense, there are two types of myocardial hypertrophy:physiological and pathological cardiomyocyte hypertrophy. Affected by different stimulating factors, cardiac structure and metabolism may change correspondingly to maintain heart pumping function. Pathological myocardial hypertrophy frequently leads to adverse consequences and eventually heart failure, while myocardial hypertrophy caused by physiological load is called physiological hypertrophy. Therefore, preventing maladaptive development of myocardial hypertrophy is critical to prevent heart failure.It is well known that the activation of the neurohumoral system contributes to the two kinds of cardiac hypertrophy. But we should keep in mind that the form of stimulation was different. Intermittent stimulation resulted in physiological cardiac hypertrophy, while continuous stimulation resulted in pathological cardiac hypertrophy. Therefore, we speculated that the different forms of the stimulation may attributable to the different types of cardiac hypertrophy. We hypothesized that the intermittent stimulation may have a protective effect on cardiac hypertrophy, while the sustained stimulation has detrimental effect. Myocardial ischemic preconditioning is a protective and adaptive phenomenon whereby brief episodes of ischemia and reperfusion render the heart resistant to subsequent ischemia. Likewise, is it possible that brief episodes of hypertrophic stimulation render the heart resistant to subsequent hypertrophic stress? Interestingly, an animal study reported that short-term antihypertensive therapy can prolong the effect of anti-myocardial-hypertrophy to protect the heart, suggesting a phenomenon of myocardial hypertrophic preconditioning. Supportively, physical training can similarly precontion the heart by intermittent activation of neurohumour factors and consequently lead to protective physiologic hypertrophy. Collectively, we propose a new concept termed "myocardial hypertrophic preconditioning." The purpose of this study is to expermentally prove the "myocardial hypertrophic preconditioning" phenomenon in both drug and pressure overload induced hypertrophy of cardiomyocyte.Methods:1. Drug-induced myocardial hypertrophyC57BL/6 male mice (8-12 wk,20-25 g) were intraperitoneally anesthetized with pentobarbital (50 mg/kg). After anesthesia, the mice were treated with phenylephrine (PE,30mg/kg/day) or vehicle by osmotic minipump (Alzet) to induce cardiac hypertrophy. After 4 days of drug-induced stimulation, the animals were sacrificed to obtain heart and calculate heart weight/body weight ratio. The mice were divided into 3 groups, with 6-7 mice in each group, as follows:(1) PE group:PE infusion for 4 days; (2) PE+PRE group:PE for 1 day, infusion stop for 1 day, PE for 2 days, infusion stop for 2 days, and than PE for 4 days; (3) Control group:ascorbic acid infusion for 4 days. 2. Transverse aortic constriction (TAC) induced myocardial hypertrophyC57BL/6 male mice (8-10 wk,18-25 g) were anesthetized with pentobarbital sodium (50mg/kg). The chest cavity was entered in the second intercostals space at the left upper sternal border through a small incision, and the thymus was then gently deflected out of the field of view to expose the aortic arch. After the transverse aorta was isolated between the carotid arteries, it was constricted by a 7-0 silk suture ligature tied firmly against a 27-gauge needle. The gauge needle was promptly removed and chest was closed. Myoardial hypertrophy and subsequent heart failure were induced.3. M-mode echocardiographyCardiac function and remodeling were dynamically evaluated with echocardiography at baseline,7,21 and 35 days. From M-mode tracing, LV end-diastolic diameter (LVEDd), LV end-systolic diameter (LVESd), diastolic and systolic LV wall thickness (Pwd, Pws) and left ventricular shortening (LVFS) were measured. LVFS= (LVEDd-LVESd)/LVEDd×100.4. PCRWhole hearts from each group were used to extract RNA. Reverse transcriptase PCR (RT-PCR) was perfomed to evaluate the expression levels of hypertrophic mark genes:atrial natriuretic peptide (ANP) and beta-myosin heavy chains ((3-MHC).5. Histological examinationsFor histological examinations hearts were fixed in 4% paraformaldehyde and embedded in paraffin 4-6μm sections. Cross section area of cardiomyocytes was calculated using Image J software.6. Experimental protocols for TAC miceExperiments were divided into two protocols,6-7 mice in each group.Protocol 1:Early window protective effect of preconditioning in pressure overload mice. Three groups were included:Sham group and TAC group, observation for 7 days; TAC+PRE group:TAC for 3 days, debanding for 4 days, then second TAC, and finally observation for 7 days.Protocol 2:Late window protective effect of preconditioning in pressure overloaded mice. Four groups were designed:Sham group and TAC group, observation for 6 weeks; TAC+PRE1 group:TAC for 3 days, debanding for 4 days, then second TAC, and finally observation for 6 weeks; TAC+PRE2 group:TAC for 1 week, debanding for 1 week, then second TAC, and finally observation for 6 weeks.Results:1. Drug-induced hypertrophic preconditioningCompared with PE group, the HW/BW ratio in PE+PRE group decreased significantly (4.02±0.04 vs.4.18±0.06 mg/g, P=0.044).2. Hypertrophic preconditioning induced by brief episode of pressure overloadIn protocol 1:compared with TAC group, the HW/BW ratio in TAC+PRE group was significantly smaller (5.99±0.22 vs.5.35±0.17 mg/g, P=0.014).In protocol 2:compared with TAC group, the HW/BW ratio in TAC+PRE1 and TAC+PRE2 groups was markedly smaller (7.16±0.33 in TAC,5.23±0.14 in TAC +PRE1, and 5.43±0.11 in TAC+PRE2, P=0.004). Compared with TAC group, the cardiomyocytes area in TAC+PRE1 group and TAC+PRE2 group decreased by about 58% and 51% respectively (609.64%±16.85% in TAC,254.83%±5.29% in TAC+ PRE1,P=0.000; and 297.76%±9.60% in TAC+PRE2, P=0.000).3. Hypertrophic preconditioning inhibited upregulation of ANP andβ-MHCIn comparison with Sham group, the expression levels of hyperophic mark genes ANP (0.91±0.09 vs.1.60±0.10, P=0.01) andβ-MHC (0.38±0.06 vs.0.93±0.06, P=0.000) were significantly up-regulated in TAC group, while hypertrophic preconditioning significantly inhibited the TAC-induced upregulation of ANP andβ-MHC, all P< 0.001).4. Hypertrophic preconditioning improves heart failureLVFS at different time points in TAC group was significantly lower compared with that in Sham group (all P≤0.001), while LVFS in TAC+PRE1 group and TAC+ PRE2 group at each time point after surgery were significantly higher than that in the TAC group (all P<0.05).Lung weight/body weight ratio in both precondionting groups was smaller than in TAC group (9.88±1.00 mg/g in TAC group,5.98±0.12 mg/g in TAC+PRE1 group, P=0.008 and 6.15±0.11 in TAC+PRE2 group, P=0.046).5. Hypertrophic preconditioning slows the progression of cardiac remodelingIn protocol 2, the M-mode echocardiographic results in different groups over time show that: in TAC group, the LVEDd and LVESd at 35 days after surgery significantly larger than those at 7 days (LVEDd:3.70±0.13 mm vs.3.15±0.07 mm, P=0.001; LVESd:2.48±0.19 mm vs.1.93±0.14 mm, P=0.025); LVEDd, LVESd, Pwd and Pws in TAC+PRE1 group and TAC+PRE2 group at 35 d after surgery were significantly lower than that in the TAC group (all P< 0.05).Conclusions:1. Precondioning by prohypertrophic factors renders the heart resistant to subsequent hypertrophic stress and delays the progression from myocardial hypertrophy to heart failure, indicting the presence of "myocardial hypertrophic preconditioning".2. Myocardial hypertrophy preconditioning exerts antihypertrophic effect, inhibits cardiac remodeling and improves heart failure.