Exercise Regulates MicroRNAs to Preserve Coronary and Cardiac Function in the Diabetic Heart.

RATIONALE: Diabetic heart disease (DHD) is a debilitating manifestation of type 2 diabetes mellitus. Exercise has been proposed as a potential therapy for DHD, although the effectiveness of exercise in preventing or reversing the progression of DHD remains controversial. Cardiac function is critically dependent on the preservation of coronary vascular function. OBJECTIVE: We aimed to elucidate the effectiveness and mechanisms by which exercise facilitates coronary and cardiac-protection during the onset and progression of DHD. METHODS AND RESULTS: Diabetic db/db and nondiabetic mice, with or without underlying cardiac dysfunction (16 and 8 weeks old, respectively) were subjected to either moderate-intensity exercise or high-intensity exercise for 8 weeks. Subsequently, synchrotron microangiography, immunohistochemistry, Western blot, and real-time polymerase chain reaction were used to assess time-dependent changes in cardiac and coronary structure and function associated with diabetes mellitus and exercise and determine whether these changes reflect the observed changes in cardiac-enriched and vascular-enriched microRNAs (miRNAs). We show that, if exercise is initiated from 8 weeks of age, both moderate-intensity exercise and high-intensity exercise prevented the onset of coronary and cardiac dysfunction, apoptosis, fibrosis, microvascular rarefaction, and disruption of miRNA signaling, as seen in the nonexercised diabetic mice. Conversely, the cardiovascular benefits of moderate-intensity exercise were absent if the exercise was initiated after the diabetic mice had already established cardiac dysfunction (ie, from 16 weeks of age). The experimental silencing or upregulation of miRNA-126 activity suggests the mechanism underpinning the cardiovascular benefits of exercise were mediated, at least in part, through tissue-specific miRNAs. CONCLUSIONS: Our findings provide the first experimental evidence for the critical importance of early exercise intervention in ameliorating the onset and progression of DHD. Our results also suggest that the beneficial effects of exercise are mediated through the normalization of cardiovascular-enriched miRNAs, which are dysregulated in DHD.

ß-blockade prevents coronary macro- and microvascular dysfunction induced by a high salt diet and insulin resistance in the Goto-Kakizaki rat.

A high salt intake exacerbates insulin resistance, evoking hypertension due to systemic perivascular inflammation, oxidative-nitrosative stress and endothelial dysfunction. Angiotensin-converting enzyme inhibitor (ACEi) and angiotensin receptor blockers (ARBs) have been shown to abolish inflammation and redox stress but only partially restore endothelial function in mesenteric vessels. We investigated whether sympatho-adrenal overactivation evokes coronary vascular dysfunction when a high salt intake is combined with insulin resistance in male Goto-Kakizaki (GK) and Wistar rats treated with two different classes of ß-blocker or vehicle, utilising synchrotron-based microangiography in vivo. Further, we examined if chronic carvedilol (CAR) treatment preserves nitric oxide (NO)-mediated coronary dilation more than metoprolol (MET). A high salt diet (6% NaCl w/w) exacerbated coronary microvessel endothelial dysfunction and NO-resistance in vehicle-treated GK rats while Wistar rats showed modest impairment. Microvascular dysfunction was associated with elevated expression of myocardial endothelin, inducible NO synthase (NOS) protein and 3-nitrotyrosine (3-NT). Both CAR and MET reduced basal coronary perfusion but restored microvessel endothelium-dependent and -independent dilation indicating a role for sympatho-adrenal overactivation in vehicle-treated rats. While MET treatment reduced myocardial nitrates, only MET treatment completely restored microvessel dilation to dobutamine (DOB) stimulation in the absence of NO and prostanoids (combined inhibition), indicating that MET restored the coronary flow reserve attributable to endothelium-derived hyperpolarisation (EDH). In conclusion, sympatho-adrenal overactivation caused by high salt intake and insulin resistance evoked coronary microvessel endothelial dysfunction and diminished NO sensitivity, which were restored by MET and CAR treatment in spite of ongoing inflammation and oxidative-nitrosative stress presumably caused by uninhibited renin-angiotensin-aldosterone system (RAAS) overactivation.

Overexpression of heart-specific small subunit of myosin light chain phosphatase results in heart failure and conduction disturbance.

Mutations in genes encoding components of the sarcomere cause cardiomyopathy, which is often associated with abnormal Ca2+ sensitivity of muscle contraction. We have previously shown that a heart-specific myosin light chain phosphatase small subunit (hHS-M21) increases the Ca2+ sensitivity of muscle contraction. The aim of the present study was to investigate the function of hHS-M21 in vivo and the causative role of abnormal Ca2+ sensitivity in cardiomyopathy. We generated transgenic mice with cardiac-specific overexpression of hHS-M21. We confirmed that hHS-M21 increased the Ca2+ sensitivity of cardiac muscle contraction in vivo, which was not followed by an increased phosphorylation of myosin light chain 2 isoforms. hHS-M21 transgenic mice developed severe systolic dysfunction with myocardial fibrosis and degeneration of cardiomyocytes in association with sinus bradycardia and atrioventricular conduction defect. The contractile dysfunction and cardiac fibrosis were improved by treatment with the Rho kinase inhibitor fasudil. Our findings suggested that the overexpression of hHS-M21 results in cardiac dysfunction and conduction disturbance via non-myosin light chain 2 phosphorylation-dependent regulation. NEW & NOTEWORTHY The present study is the first to develop mice with transgenic overexpression of a heart-specific myosin light chain phosphatase small subunit (hHS-M21) and to examine the effects of hHS-M21 on cardiac function. Elevation of hHS-M21 induced heart failure with myocardial fibrosis and degeneration of cardiomyocytes accompanied by supraventricular arrhythmias.

Myocardial interstitial levels of serotonin and its major metabolite 5-hydroxyindole acetic acid during ischemia-reperfusion.

The aim of this study was to examine the accumulation of serotonin (5-HT) and degradation of 5-HT taken up into cells in the ischemic region during myocardial ischemia-reperfusion. Using microdialysis technique in anesthetized rats, we monitored myocardial interstitial levels of 5-HT and its metabolite produced by monoamine oxidase (MAO), 5-hydroxyindole acetic acid (5-HIAA), during 30-min coronary occlusion followed by 45-min reperfusion, and investigated the effects of local administration of the MAO inhibitor pargyline and the 5-HT uptake inhibitor fluoxetine. In the vehicle group, the dialysate 5-HT concentration increased from 1.3 ± 0.2 nM at baseline to 29.6 ± 2.8 nM at 22.5-30 min of occlusion, but the dialysate 5-HIAA concentration did not change from baseline (9.9 ± 1.1 nM). Upon reperfusion, the dialysate 5-HT concentration increased further to a peak (34.2 ± 4.2 nM) at 0-7.5 min and then declined. The dialysate 5-HIAA concentration increased to 31.9 ± 5.2 nM at 7.5-15 min of reperfusion and maintained this high level until 45 min. Pargyline markedly suppressed the increase in dialysate 5-HIAA concentration after reperfusion and increased the averaged dialysate 5-HT concentration during the reperfusion period. Fluoxetine suppressed the increase in dialysate 5-HT concentration during occlusion but did not change dialysate 5-HT or 5-HIAA concentration after reperfusion. During ischemia, 5-HT secreted from ischemic tissues accumulates but 5-HT degradation by MAO is suppressed. After reperfusion, degradation of 5-HT taken up into cells is enhanced and contributes to the clearance of accumulated 5-HT. This degradation following cellular uptake is dependent on MAO activity but not the fluoxetine-sensitive uptake transporter. NEW & NOTEWORTHY: By monitoring myocardial interstitial levels of 5-HT and its metabolite, 5-hydroxyindole acetic acid, we investigated 5-HT kinetics during myocardial ischemia-reperfusion. 5-HT accumulates but 5-HT degradation is suppressed during ischemia. After reperfusion, 5-HT degradation is enhanced and this degradation is dependent on monoamine oxidase activity but not fluoxetine-sensitive uptake transporter.

GSK-3ß heterozygous knockout is cardioprotective in a knockin mouse model of familial dilated cardiomyopathy.

Glycogen synthase kinase-3ß (GSK-3ß) plays a central role in both cardiac physiology and pathology. Herein we want to clarify the role of GSK-3ß in familial dilated cardiomyopathy. We generated a mouse model carrying a heterozygous knockout mutation of GSK-3ß (GSK-3ß(+/-) KO), together with a ΔK210 knockin mutation in cardiac troponin T (ΔK210 cTnT KI), which was proved to be one of the genetic causes of familial dilated cardiomyopathy (DCM). GSK-3ß(+/-) KO prevented the slow and rapid deterioration in left ventricular systolic function accompanying heart failure (HF) in DCM mice with heterozygous and homozygous ΔK210 cTnT KI mutations, respectively. GSK-3ß(+/-) KO also prevented cardiac enlargement, myocardial fibrosis, and cardiomyocyte apoptosis and markedly reduced the expression of cardiac ß-myosin heavy chain isoform, indicative of HF, in DCM mice with homozygous ΔK210 cTnT KI mutation. GSK-3ß(+/-) KO also extended the life span of these DCM mice. This study suggests that the inhibition of GSK-3ß is cardioprotective in familial DCM associated with ΔK210 cTnT mutation.

Chronic Rho-kinase inhibition improves left ventricular contractile dysfunction in early type-1 diabetes by increasing myosin cross-bridge extension.

BACKGROUND: Impaired actin-myosin cross-bridge (CB) dynamics correlate with impaired left ventricular (LV) function in early diabetic cardiomyopathy (DCM). Elevated expression and activity of Rho kinase (ROCK) contributes to the development of DCM. ROCK targets several sarcomeric proteins including myosin light chain 2, myosin binding protein-C (MyBP-C), troponin I (TnI) and troponin T that all have important roles in regulating CB dynamics and contractility of the myocardium. Our aim was to examine if chronic ROCK inhibition prevents impaired CB dynamics and LV dysfunction in a rat model of early diabetes, and whether these changes are associated with changes in myofilament phosphorylation state. METHODS: Seven days post-diabetes induction (65 mg/kg ip, streptozotocin), diabetic rats received the ROCK inhibitor, fasudil (10 mg/kg/day ip) or vehicle for 14 days. Rats underwent cardiac catheterization to assess LV function simultaneous with X-ray diffraction using synchrotron radiation to assess in situ CB dynamics. RESULTS: Compared to controls, diabetic rats developed mild systolic and diastolic dysfunction, which was attenuated by fasudil. End-diastolic and systolic myosin proximity to actin filaments were significantly reduced in diabetic rats (P < 0.05). In all rats there was an inverse correlation between ROCK1 expression and the extension of myosin CB in diastole, with the lowest ROCK expression in control and fasudil-treated diabetic rats. In diabetic and fasudil-treated diabetic rats changes in relative phosphorylation of TnI and MyBP-C were not significant from controls. CONCLUSIONS: Our results demonstrate a clear role for ROCK in the development of LV dysfunction and impaired CB dynamics in early DCM.

Role of brain serotonin dysfunction in the pathophysiology of congestive heart failure.

Inherited or non-inherited dilated cardiomyopathy (DCM) patients develop varied disease phenotypes leading to death after developing congestive heart failure (HF) or sudden death with mild or no overt HF symptoms, suggesting that environmental and/or genetic factors may modify the disease phenotype of DCM. In this study, we sought to explore unknown genetic factors affecting the disease phenotype of monogenic inherited human DCM. Knock-in mice bearing a sarcomeric protein mutation that causes DCM were created on different genetic backgrounds; BALB/c and C57Bl/6. DCM mice on the BALB/c background showed cardiac enlargement and systolic dysfunction and developed congestive HF before died. In contrast, DCM mice on the C57Bl/6 background developed no overt HF symptoms and died suddenly, although they showed considerable cardiac enlargement and systolic dysfunction. BALB/c mice have brain serotonin dysfunction due to a single nucleotide polymorphism (SNP) in tryptophan hydroxylase 2 (TPH2). Brain serotonin dysfunction plays a critical role in depression and anxiety and BALB/c mice exhibit depression- and anxiety-related behaviors. Since depression is common and associated with poor prognosis in HF patients, we examined therapeutic effects of anti-depression drug paroxetine and anti-anxiety drug buspirone that could improve the brain serotonin function in mice. Both drugs reduced cardiac enlargement and improved systolic dysfunction and symptoms of severe congestive HF in DCM mice on the BALB/c background. These results strongly suggest that genetic backgrounds involving brain serotonin dysfunction, such as TPH2 gene SNP, may play an important role in the development of congestive HF in DCM.

ß-Arrestin-Biased AT1 Agonist TRV027 Causes a Neonatal-Specific Sustained Positive Inotropic Effect Without Increasing Heart Rate.

The treatment of pediatric heart failure is a long-standing unmet medical need. Angiotensin II supports mammalian perinatal circulation by activating cardiac L-type Ca2+ channels through angiotensin type 1 receptor (AT1R) and ß-arrestin. TRV027, a ß-arrestin-biased AT1R agonist, that has been reported to be safe but not effective for adult patients with heart failure, activates the AT1R/ß-arrestin pathway. We found that TRV027 evokes a long-acting positive inotropic effect specifically on immature cardiac myocytes through the AT1R/ß-arrestin/L-type Ca2+ channel pathway with minimum effect on heart rate, oxygen consumption, reactive oxygen species production, and aldosterone secretion. Thus, TRV027 could be utilized as a valuable drug specific for pediatric heart failure.

Knock-in mouse model of dilated cardiomyopathy caused by troponin mutation.

We created knock-in mice in which a deletion of 3 base pairs coding for K210 in cardiac troponin (cTn)T found in familial dilated cardiomyopathy patients was introduced into endogenous genes. Membrane-permeabilized cardiac muscle fibers from mutant mice showed significantly lower Ca(2+) sensitivity in force generation than those from wild-type mice. Peak amplitude of Ca(2+) transient in cardiomyocytes was increased in mutant mice, and maximum isometric force produced by intact cardiac muscle fibers of mutant mice was not significantly different from that of wild-type mice, suggesting that Ca(2+) transient was augmented to compensate for decreased myofilament Ca(2+) sensitivity. Nevertheless, mutant mice developed marked cardiac enlargement, heart failure, and frequent sudden death recapitulating the phenotypes of dilated cardiomyopathy patients, indicating that global functional defect of the heart attributable to decreased myofilament Ca(2+) sensitivity could not be fully compensated by only increasing the intracellular Ca(2+) transient. We found that a positive inotropic agent, pimobendan, which directly increases myofilament Ca(2+) sensitivity, had profound effects of preventing cardiac enlargement, heart failure, and sudden death. These results verify the hypothesis that Ca(2+) desensitization of cardiac myofilament is the absolute cause of the pathogenesis of dilated cardiomyopathy associated with this mutation and strongly suggest that Ca(2+) sensitizers are beneficial for the treatment of dilated cardiomyopathy patients affected by sarcomeric regulatory protein mutations.

Identification and physiological activity of survival factor released from cardiomyocytes during ischaemia and reperfusion.

AIMS: We carried out a screening of survival factors released from cells exposed to simulated ischaemia and reperfusion (sI/R) using the embryonic rat heart-derived cell line, H9c2 cells, and examined the physiological role of the identified factor. METHOD AND RESULTS: The culture medium supernatant of H9c2 cells exposed to sI/R was separated by column chromatography and the fractions examined for survival activity. The protein with survival activity was identified by mass spectrometry, and its physiological role was examined in the models of ischaemia. Cell survival activity was detected in at least three fractions of the cell supernatant collected during sI/R and subjected to a series of column chromatographic steps. Among the proteins measured by mass spectrometry and western blotting, a p36 protein identified as a glycolytic enzyme, lactate dehydrogenase muscle subunit (M-LDH), showed strong survival activity. H(2)O(2)-induced intracellular calcium overload in H9c2 cells and irregular Ca(2+) transients in adult rat cardiomyocytes were both found to be inhibited by pretreatment with M-LDH. M-LDH also lowered the frequency and amplitude of early afterdepolarizations induced by H(2)O(2) in adult rat cardiomyocytes and suppressed the ischaemia-reperfusion-induced reduction of cardiac output from mouse working heart preparations. M-LDH was found to increase the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), which plays a role in H9c2 cell survival. CONCLUSION: M-LDH released from cardiomyocytes after hypoxia and reoxygenation has a role in protecting the heart from oxidative stress-induced injury through an intracellular signal transduction pathway involving ERK1/2.

Serotonin uptake via plasma membrane monoamine transporter during myocardial ischemia-reperfusion in the rat heart in vivo.

Serotonin (5-HT) accumulates in the heart during myocardial ischemia and induces deleterious effects on the cardiomyocytes through receptor-dependent and monoamine oxidase-dependent pathways. We aimed to clarify the involvement of extra-neuronal monoamine transporters in the clearance of 5-HT during ischemia and reperfusion in the heart. Using a microdialysis technique in the anesthetized Wistar rat heart, we monitored myocardial interstitial 5-HT and 5-hydroxyindole acetic acid (5-HIAA) concentration by means of electro-chemical detection coupled with high-performance liquid chromatography (HPLC-ECD). Effects of inhibitors of the plasma membrane monoamine transporter (PMAT) and the organic cation transporter 3 (OCT3) (decynium-22 and corticosterone) on the 5-HT and 5-HIAA concentrations during baseline, coronary occlusion, and reperfusion were investigated. Basal dialysate 5-HT concentration were increased by local administration of decynium-22, but not by corticosterone. Addition of fluoxetine, a serotonin transporter (SERT) inhibitor further increased the 5-HT concentration upon during administration of decynium-22. Decynium-22 elevated the background level of 5-HT during coronary occlusion and maintained 5-HT concentration at a high level during reperfusion. Production of 5-HIAA in the early reperfusion was significantly suppressed by decynium-22. These results indicate that PMAT and SERT independently regulate basal level of interstitial 5-HT, and PMAT plays a more important role in the clearance of 5-HT during reperfusion. These data suggest the involvement of PMAT in the monoamine oxidase-dependent deleterious pathway in the heart.

Ghrelin and vascular protection.

Ghrelin is a small peptide with important roles in the regulation of appetite, gut motility, glucose homeostasis as well as cardiovascular protection. This review highlights the role that acyl ghrelin plays in maintaining normal endothelial function by maintaining the balance of vasodilator-vasoconstrictor factors, inhibiting inflammatory cytokine production and immune cell recruitment to sites of vascular injury and by promoting angiogenesis.

Progressive Decrease in Coronary Vascular Function Associated With Type 2 Diabetic Heart Disease.

Background: The causal factors underpinning the onset and progression of diabetic heart disease (DHD) remain to be fully elucidated. Myocardial function is critically dependent on optimal coronary blood flow. Considering vascular disease occurs early in diabetes due to endothelial dysfunction, this study aimed to determine whether impaired coronary perfusion contributes to the origins of myocardial dysfunction in DHD, or whether coronary and cardiac dysfunction are independent pathologies associated with diabetes. Methods: Synchrotron radiation microangiography was used to image the coronary circulation of type-2 diabetic db/db and non-diabetic db/+ mice in vivo at 8, 16, and 24 weeks of age. We further assessed vascular function based on the vasodilatory responses to acetylcholine (ACh, 3 µg/kg/min), sodium nitroprusside (SNP, 5 µg/kg/min) and the Rho-kinase inhibitor, fasudil (20 mg/kg, i.v.). Cardiac function was assessed using echocardiography, and cardiac eNOS and ROCK expression were measured using immunohistochemistry. Results: Coronary and cardiac function were normal in 8-week-old diabetic mice. However, by 16 weeks of age, diabetic mice had advanced cardiac dysfunction. In comparison, normal coronary perfusion was preserved in diabetes until 24 weeks of age. Moreover, only the 24-week-old diabetic mice showed clear evidence of advanced coronary vascular dysfunction, based on (i) the absence of a vasodilatory response to ACh, and (ii) an exaggerated vasodilatory response to fasudil. Interestingly, fasudil also restored normal coronary perfusion in the 24-week-old diabetic heart by restoring blood flow to previously constricted vessels (diameter < 100 µm). Importantly, there was a ubiquitous decrease, and increase, in the cardiac expression of eNOS and ROCK, respectively. Conclusion: These results suggest that both cardiac and coronary dysfunction appear to have independent origins associated with diabetes and Rho-kinase pathway may be playing a role in the onset and progression of DHD.

Cardiac vagal control in a knock-in mouse model of dilated cardiomyopathy with a troponin mutation.

The aim of this study was to evaluate cardiac vagal nerve activity and identify the abnormality of cardiac vagal control in heart failure caused by dilated cardiomyopathy (DCM) using a knock-in mouse model with a ΔK210 mutation in the cardiac troponin T gene. The effects of electrical stimulation of the cervical vagal nerve at 5 and 10Hz (peripheral vagal control) and α2-adrennoceptor stimulation by intravenous medetomidine at 0.1mg/kg (central vagal control) were examined in wild-type (WT) mice and DCM mice. Microdialysis technique was applied to the left ventricular myocardium of anesthetized mice and myocardial interstitial acetylcholine (ACh) levels were measured by HPLC as an index of ACh release from cardiac vagal nerve endings. Electrical vagal nerve stimulation increased cardiac interval and myocardial interstitial ACh level in both WT and DCM mice, and these responses did not differ between WT and DCM mice. In contrast, intravenous medetomidine increased cardiac interval and myocardial interstitial ACh level in both WT and DCM mice, but the responses of cardiac interval and myocardial interstitial ACh level were significantly suppressed in DCM mice compared to WT mice. Medetomidine did not affect the myocardial interstitial ACh response induced by vagal nerve stimulation in WT mice. In this mouse model of DCM, peripheral vagal control including ACh release from vagal nerve endings and the postsynaptic response of pacemaker cells was preserved, but central vagal control through α2-adrenoceptors was impaired.

Monoamine oxidase-induced hydroxyl radical production and cardiomyocyte injury during myocardial ischemia-reperfusion in rats.

To elucidate the involvement of monoamine oxidase (MAO) in hydroxyl radical production and cardiomyocyte injury during ischemia as well as after reperfusion, we applied microdialysis technique to the heart of anesthetized rats. Dialysate samples were collected during 30 min of induced ischemia followed by 60 min of reperfusion. We monitored dialysate 3,4-dihydrobenzoic acid (3,4-DHBA) concentration as an index of hydroxyl radical production using a trapping agent (4-hydroxybenzoic acid), and dialysate myoglobin concentration as an index of cardiomyocyte injury in the ischemic region. The effect of local administration of a MAO inhibitor, pargyline, was investigated. Dialysate 3,4-DHBA concentration increased from 1.9 ± 0.5 nM at baseline to 3.5 ± 0.7 nM at 20-30 min of occlusion. After reperfusion, dialysate 3,4-DHBA concentration further increased reaching a maximum (4.5 ± 0.3 nM) at 20-30 min after reperfusion, and stabilized thereafter. Pargyline suppressed the averaged increase in dialysate 3,4-DHBA concentration by ∼72% during occlusion and by ∼67% during reperfusion. Dialysate myoglobin concentration increased from 235 ± 60 ng/ml at baseline to 1309 ± 298 ng/ml at 20-30 min after occlusion. After reperfusion, dialysate myoglobin concentration further increased reaching a peak (5833 ± 1017 ng/ml) at 10-20 min after reperfusion, and then declined. Pargyline reduced the averaged dialysate myoglobin concentration by ∼56% during occlusion and by ∼41% during reperfusion. MAO plays a significant role in hydroxyl radical production and cardiomyocyte injury during ischemia as well as after reperfusion.

In vivo effects of propyl gallate, a novel Ca(2+) sensitizer, in a mouse model of dilated cardiomyopathy caused by cardiac troponin T mutation.

AIMS: We have previously demonstrated that propyl gallate has a Ca(2+) sensitizing effect on the force generation in membrane-permeabilized (skinned) cardiac muscle fibers. However, in vivo beneficial effects of propyl gallate as a novel Ca(2+) sensitizer remain uncertain. In the present study, we aim to explore in vivo effects of propyl gallate. MAIN METHODS: We compared effects of propyl gallate on ex vivo intact cardiac muscle fibers and in vivo hearts in healthy mice with those of pimobendan, a clinically used Ca(2+) sensitizer. The therapeutic effect of propyl gallate was investigated using a mouse model of dilated cardiomyopathy (DCM) with reduced myofilament Ca(2+) sensitivity due to a deletion mutation ΔK210 in cardiac troponin T. KEY FINDINGS: Propyl gallate, as well as pimobendan, showed a positive inotropic effect. Propyl gallate slightly increased the blood pressure without changing the heart rate in healthy mice, whereas pimobendan decreased the blood pressure probably through vasodilation via inhibition of phosphodiesterase and increased the heart rate. Propyl gallate prevented cardiac remodeling and systolic dysfunction and significantly improved the life-expectancy of knock-in mouse model of DCM with reduced myofilament Ca(2+) sensitivity due to a mutation in cardiac troponin T. On the other hand, gallate, a similarly strong antioxidant polyphenol lacking Ca(2+) sensitizing action, had no beneficial effects on the DCM mice. SIGNIFICANCE: These results suggest that propyl gallate might be useful for the treatment of inherited DCM caused by a reduction in the myofilament Ca(2+) sensitivity.

Myocardial interstitial serotonin and its major metabolite, 5-hydroxyindole acetic acid levels determined by microdialysis technique in rat heart.

AIMS: The aim of this study was to elucidate myocardial interstitial serotonin (5-HT) kinetics in the heart, including 5-HT reuptake and enzymatic degradation to 5-hydroxyindole acetic acid (5-HIAA) via monoamine oxidase (MAO). MAIN METHODS: Using microdialysis technique in anesthetized rats, we simultaneously monitored myocardial interstitial levels of 5-HT and its major metabolite, 5-HIAA, in the left ventricle and examined the effects of local administration of a MAO inhibitor, pargyline, or a 5-HT uptake inhibitor, fluoxetine. KEY FINDINGS: Pargyline increased dialysate 5-HT concentration from 1.8±0.3 at baseline to 3.9±0.5nM but decreased dialysate 5-HIAA concentration from 20.7±1.0 at baseline to 15.8±1.4nM at 60-80min of administration. Fluoxetine increased dialysate 5-HT concentration from 1.9±0.4 at baseline to 6.5±0.9nM at 60-80min of administration, but did not change dialysate 5-HIAA concentration. Local administration of ADP (100mM) increased dialysate 5-HT and 5-HIAA concentrations. Pargyline did not affect ADP-induced increase in dialysate 5-HT concentration but suppressed ADP-induced increase in dialysate 5-HIAA concentration during 60min of ADP administration. Fluoxetine increased dialysate 5-HT concentration at 40-60min of ADP administration, but did not affect ADP-induced increase in dialysate 5-HIAA concentration. SIGNIFICANCE: Simultaneous monitoring of myocardial interstitial 5-HT and 5-HIAA levels provides valuable information on 5-HT kinetics including reuptake and enzymatic degradation by MAO, which play a role in the regulation of myocardial interstitial 5-HT levels at baseline and when 5-HT levels are elevated.

Survival benefit of ghrelin in the heart failure due to dilated cardiomyopathy.

Although ghrelin has been demonstrated to improve cardiac function in heart failure, its therapeutic efficacy on the life expectancy remains unknown. We aim to examine whether ghrelin can improve the life survival in heart failure using a mouse model of inherited dilated cardiomyopathy (DCM) caused by a deletion mutation ΔK210 in cardiac troponin T (cTnT). From 30 days of age, ghrelin (150 µg/kg) was administered subcutaneously to DCM mice once daily, control mice received saline only. The survival rates were compared between the two groups for 30 days. After 30-day treatment, functional and morphological measurements were conducted. Ghrelin-treated DCM mice had significantly prolonged life spans compared with saline-treated control DCM mice. Echocardiography showed that ghrelin reduced left ventricular (LV) end-diastolic dimensions and increased LV ejection fraction. Moreover, histoanatomical data revealed that ghrelin decreased the heart-to-body weight ratio, prevented cardiac remodeling and fibrosis, and markedly decreased the expression of brain natriuretic peptide. Telemetry recording and heart rate variability analysis showed that ghrelin suppressed the excessive cardiac sympathetic nerve activity (CSNA) and recovered the cardiac parasympathetic nerve activity. These results suggest that ghrelin has therapeutic benefits for survival as well as for the cardiac function and remodeling in heart failure probably through suppression of CSNA and recovery of cardiac parasympathetic nerve activity.

In vivo monitoring of acetylcholine release from cardiac vagal nerve endings in anesthetized mice.

We applied a microdialysis technique to the left ventricular myocardium of anesthetized mice and tried to monitor acetylcholine (ACh) release from cardiac vagal nerves. Transection of bilateral cervical vagal nerves decreased dialysate ACh concentration. Electrical stimulation of the left cervical vagal nerve increased dialysate ACh concentration in proportion to the frequency of stimulation. Intravenous administration of hexamethonium, prevented the increase in dialysate ACh concentration during vagal nerve stimulation, indicating that ACh in the dialysate primarily reflects ACh released from post-ganglionic cardiac vagal nerves. Microdialysis permits monitoring of ACh release from post-ganglionic cardiac vagal nerves that are most likely to be innervating the left ventricle in mice.

Up-regulation of type 2 iodothyronine deiodinase in dilated cardiomyopathy.

AIMS: Thyroid hormone (TH) has prominent effects on the heart, and hyperthyroidism is occasionally found to be a cause of dilated cardiomyopathy (DCM). We aim to explore the potential role of TH in the pathogenesis of DCM. METHODS AND RESULTS: The pathophysiological role of TH in the heart was investigated using a knock-in mouse model of inherited DCM with a deletion mutation DeltaK210 in the cardiac troponin T gene. Serum tri-iodothyronine (T(3)) levels showed no significant difference between wild-type (WT) and DCM mice, whereas cardiac T(3) levels in DCM mice were significantly higher than those in WT mice. Type 2 iodothyronine deiodinase (Dio2), which produces T(3) from thyroxin, was up-regulated in the DCM mice hearts. The cAMP levels were increased in DCM mice hearts, suggesting that transcriptional up-regulation of Dio2 gene is mediated through the evolutionarily conserved cAMP-response element site in its promoter. Propylthiouracil (PTU), an anti-thyroid drug, prevented the hypertrophic remodelling of the heart in DCM mice and improved their cardiac function and life expectancy. Akt and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation increased in the DCM mice hearts and PTU treatment significantly reduced the phosphorylation levels, strongly suggesting that Dio2 up-regulation is involved in cardiac remodelling in DCM through activating the TH-signalling pathways involving Akt and p38 MAPK. Dio2 gene expression was also markedly up-regulated in the mice hearts developing similar eccentric hypertrophy after myocardial infarction. CONCLUSION: Local hyperthyroidism via transcriptional up-regulation of the Dio2 gene may be an important underlying mechanism for the hypertrophic cardiac remodelling in DCM.