The Mechanism Of Cardiac Dysfunction In Burn And Muscular Dystrophy

Background and Aims:Multiple organ dysfunction symdrome (MODS) is one of the three major causes ofmortality after burn injury. As the heart is essential for maintain normal function of allorgans, cardiac injury after burn not only causes cardiac dysfunction, but also ischemia andhypoxia of other vital tissues. In the clinic, after a severe burn, cardiac dysfunction appearsearly and may induce or exacerbate shock. This is one of the important facets of MODSafter burn. Basic research has revealed the underlying mechanisms for post burn cardiacdysfunction including:1. As a strong stress, burn causes the activation of sympathoadrenergic system andmarked elevation of blood catecholamine concentration, which persistently stimulateβ-adrenergic receptors on the cardiac myocytes and induces cardiac myocyte death.2. There is a large amount of exudate from the burned area causing low bloodvolume，which could cause global ischemia of the heart and cardiomyocyte apoptosis.3. The barrier function of the skin and the gut is impaired and infection on a largebody surface area and the gut may ensue. Many systemic or local inflammatory factorsreach the heart and exert an inhibitory effect on the contractile function of cardiomyocytes.Prostaglandin E2(PGE2) is an important factor regulating cellular function andgrowth. It widely exists in many organs including the heart. It is a metabolite of arachidonicacid catalyzed by cyclooxygenase (COX). It is unsaturated20-carbon fatty acid. As one ofthe prostaglandins, it exerts effects including vascular dilation to decrease vascularresistance and blood pressure while increase organ blood flow, and bronchial smoothmuscle relaxation to reduce ventilation resistance. Interestingly, PGE2exerts bothinflammation initiation/promotion and immunosuppression/anti-inflammation effects. EP2and EP4, the two PGE2receptors are expressed on the cardiac myocyte. PGE2stimulation on these two receptors causes increases in intracellular cAMP.As part of the stress response, when the organism is challenged by harmful stimulisuch as hypoxia, burn and trauma, the sympathoadrenergic system is excited to releasecatecholamines leading to an increase of catecholamine concentration in the blood.Catecholamines binds to and activates β-adrenergic receptors (β AR) to activate proteinkinase A via the Gs-adenylyl cyclase (AC)-cAMP signaling pathway. PKA phosphorylatesmultiple proteins and alter their functions to make the heart adapt to the new challenges.As two important members of the molecular signals mediated by G-protein coupledreceptors, PGE2and catecholamine have their well-defined signaling pathways. However,it is still unclear if and how PGE2regulates cardiac function under stressed conditions suchas inflammation. This study aims to determine the progression, molecular and cellularmechanisms of cardiac dysfunction induced by severe burn and Duchenn musculardystrophy; and reveal that how PGE2regulates cardiac function and cardiac response toβ-adrenergic stimulation. Our study will suggest a potential role of PGE2in cardiacdysfunction induced by inflammation and provide the basics for using PGE2as a potentialtarget for treating cardiac dysfunction after severe burn or inflammation.Methods：Part I: Cardioprotection by mesenteric lymph duct ligation after burnThis study established a burn rat model and used mesenteric lymph ligation to preventthe lymph from the intestine to enter the blood stream. Then we determined early cardiacdysfunction at3hours after burn. Specifically, a30%TBSA scald rat model was prepared.We chose3hours after scalding as the experiment time point. Then rats were divided intothree experimental groups as follows:(1) control rats (without burn);(2)30%TBSA scaldrats;(3)30%TBSA scald rats with mesenteric lymph ligation surgery. To evaluate leftventricle function, we measured left ventricle systolic pressure (LVSP), left ventricle enddiastolic pressure (LVEDP), developed pressure, maximum+dp/dt, minimum-dp/dt andtime to dp/dt max.Part II: Chronic inflammation blunts cardiac β-adrenergic response in mdx miceWe use mdx mouse (a muscular dystrophy model) to mimic long-term chronicsystemic inflammation stimulation after burn. 1. We used echocardiography to observe basal cardiac function of control and mdxmice from2month old to12month old; used Masson’s trichrome stainig to determinefibrosis.2. We used in vivo hemodynamics to observe cardiac function at baseline and afterstimulation with isoproterenol (a β-adrenergic agonist) of control and mdx mice from2month old to12month old.3. We used Langendorff-perfusion heart preparation, freshly isolated cardiomyocytesand Western blot to explain the reduced β-adrenergic response of mdx hearts at the organ,cell and molecule levels.Part III: PGE2inhibits contraction of heart caused by β-adrenergic receptoractivation1. Used Langendorff perfusion heart preparation to determine the effects of differentdose of PGE2on mouse heart contractile function.2. Used Langendorff perfusion heart preparation to test the response of normal mouseheart to isoproterenol with or without0.1μM PGE2pretreatment.3. Measured the effect of0.1μM PGE2on myocyte contraction and Ca2+transientwith or without10μM rolipram treatment.4. Measured the response of myocyte contraction and Ca2+transient to isoproterenolwith or without0.1μM PGE2pretreatment.5. Measured the phosphorylation level of PDE4isoforms with Western blot.Results：Part I: Cardioprotection by mesenteric lymph duct ligation after burn1. Severe burn probably causes acute inflammation and inflammatory factors from themensenteric lymph may enter the blood stream to inhibit cardiac function. Cardiac functiondepression appears as early as3hours.2. Blocking lymph entrance into the blood stream by mesenteric lymph duct ligationprotected hearts from function depression.Part II: Chronic inflammation blunts cardiac β-adrenergic response in mdx mice 1. Loss of cardiomyocytes in mdx hearts was gradually increased with age andcardiac fibrosis was found as early as at the age of2months. However, cardiac dysfunctionin mdx mice was found only after the age of8months.2. A reduction of cardiac-adrenergic response to isoproterenol was found in mdxmice since a young age (4months) in vivo or in vitro (isolated hearts).3. Four-and8-month mdx ventricular myocytes (VMs) had greater baseline function(fractional shortening,[Ca2+]i, and SR Ca2+content) and ICa-Lthan age-matched controlVMs and2-month mdx myocytes but had reduced response to isoproterenol.4. No change of the abundance of Cav1.21c, Cav1.21, Cav1.22, SERCA,Na+/Ca2+exchange but increased total RyR2and basal phosphorylation of RyR2,phospholamban, and Cav1.21c were found in4-month mdx hearts due to increasedbaseline PKA activity. Isoproterenol increased their phosphorylation to the same level inmdx and control hearts.5. Four-month mdx VMs had reduced1-adrenergic receptor density andβ-adrenergic sensitivity.Part III: PGE2inhibits contraction of heart caused by β-adrenergic receptoractivation1.0.1uM PGE2had minimal effect on cardiac function and only higherconcentrations of PGE2enhanced cardiac function.2. Pretreatment with0.1μMPGE2inhibited cardiac ISO response.3. PGE2at0.1uM, did not increase myocyte fractional shortening and Ca2+transientamplitudes, however, in the presence of rolipram (10uM), PGE2increased myocytefunction.4. PGE2at0.1uM blocks myocyte AR responses. Myocytes not pretreated hadnormal ISO responses but PGE2pretreatment eliminated myocyte ISO responses. Thisinhibition of AR responses of myocytes is reversed by a PDE4inhibitor, rolipram (10uM).5. Inhibition of PDE4abolishes the inhibitory effect of PGE2on the adrenergicsignaling for myocyte contractile response.ConclusionThis study explored cardiac dysfunction and its underlying molecular and cellular mechanisms induced by severe burn and muscular dystrophy. A potential role ofinflammatory factor PGE2in cardiac dysfunction under these conditions was sought.Based on our results, we conclude:1. At the early stage, severe burn suppresses cardiac function through factors in themensenteric lymph that enter the blood stream; chronic inflammation induced by musculardystrophy inhibits cardiac-adrenergic response.2. PGE2as an important inflammatory factor, its concentration is marked elevated inthe blood after severe burn or muscular dystrophy. PGE2can moderately increaseintracellular cAMP in cardiac myocytes but this cAMP is not sufficient to enhance cardiaccontractility directly. However, it may activate PDE4D to limit the perfusion of cAMPinduced by-adrenergic activation and thus reduce cardiac and myocyte-adrenergicresponse.