Yasuki Kihara

Direct measurement of changes in intracellular calcium transients during hypoxia, ischemia, and reperfusion of the intact mammalian heart.

In studies of ischemia and reperfusion, a major experimental problem has been the inability to measure intracellular ionized calcium ([Ca2+])) in the intact heart. We have developed a new approach in which the bioluminescent calcium indicator aequorin is used to measure [Ca2+]i, in the isolated, coronary-perfused ferret heart. Aequorin is loaded into subepicardial myocytes of the left ventricle, and the signals are recorded simultaneously along with isovolumic left ventricular (LV) pressure at a constant pacing rate. This system shows 1) no attenuation or change of time course of LV pressure development or coronary perfusion pressure after aequorin loading; 2) consistent responses to physiological interventions and drugs; 3) individual aequorin and pressure signals that do not require signal averaging for analysis; and 4) [Ca2+]i, levels comparable with those reported in tissue or isolated myocyte cell preparations. During 5 minutes of hypoxia, diastolic [Ca2+)i and LV diastolic pressure increased while the systolic values of both [Ca2+]i and pressure decreased. The peak-to-peak systolic [Ca2+]i versus LV isovolumic pressure relation remained close to the control curve. In contrast, during 3 minutes of global ischemia, LV systolic and diastolic pressures fell rapidly, while [Ca2+]i increased substantially. The [Ca2+]i versus pressure relations for both systole and diastole shifted to the right, indicating desensitization of the contractile apparatus to [Ca2+]i. These results provide evidence that different primary mechanisms determine the systolic and diastolic responses to acute hypoxia versus ischemia. During hypoxia, changes in [Ca2+]i handling probably play a major role, while during ischemia, changes in the Ca2+ sensitivity of the myofilaments appear to be of primary importance in the modulation of contractile dysfunction.

Increased Expression of Interleukin-1β and Monocyte Chemotactic and Activating Factor/Monocyte Chemoattractant Protein-1 in the Hypertrophied and Failing Heart With Pressure Overload

Studies on the effects of proinflammatory cytokines on the heart suggest that they play some roles in the pathogenesis of congestive heart failure (CHF). To determine the involvement of proinflammatory cytokine in cardiac hypertrophy and CHF induced by mechanical overload, we investigated the expression of interleukin (IL)-1 beta and monocyte chemotactic and activating factor (MCAF)/monocyte chemoattractant protein-1 (MCP-1) in the left ventricle (LV) of Dahl salt-sensitive (DS) rats that showed hypertrophy of the LV induced by hypertension and subsequently developed CHF. The IL-1 beta mRNA content in the LV of DS rats increased 3.9-fold when LV hypertrophy developed, and the increase reached 6.2-fold at the CHF stage compared with that of age-matched Dahl salt-resistant (DR) rats. The amount of IL-1 beta in the LV was positively correlated with the LV weight/body weight ratio. Most of the IL-1 beta immunoreactivity was localized in the endothelial cells and interstitial macrophages. The mRNA levels of MCAF in the LV increased 3.6-fold at 11 weeks and reached 4.8-fold at the CHF stage relative to the age-matched DR rats. MCAF protein was localized to the endothelial cells and interstitial macrophages. In DS rats, the number of interstitial macrophages increased diffusely throughout the LV. We suggest that increased chemokine expression, macrophage infiltration, and proinflammatory cytokine expression play some role in the pathogenesis of cardiac hypertrophy and failure induced by chronic mechanical overload.

Intramuscular Transplantation of G‐CSF‐Mobilized CD34+ Cells in Patients With Critical Limb Ischemia: A Phase I/IIa, Multicenter, Single‐Blinded, Dose‐Escalation Clinical Trial

A number of preclinical studies have indicated the therapeutic potential of endothelial progenitor cells for vascular regeneration in ischemic diseases. A phase I/IIa clinical trial of transplantation of autologous CD34+ cells, the endothelial and hematopoietic progenitor‐enriched fraction, was performed in no‐option patients with atherosclerotic peripheral artery disease or Buergers disease with critical limb ischemia (CLI). CD34+ cells were isolated from the G‐CSF‐mobilized apheresis product using a magnetic cell sorting system. CD34+ cells (105/kg, n = 6; 5 × 105/kg, n = 8; or 106/kg, n = 3) were injected i.m. into the leg with more severe ischemia. The Efficacy Score, representing changes in the toe brachial pressure index (TBPI), Wong‐Baker FACES pain rating scale, and total walking distance 12 weeks after cell transplantation, the primary endpoint, was positive, indicating improvement in limb ischemia in all patients, although no significant dose‐response relationship was observed. During the 12‐week observation after cell therapy, the Wong‐Baker FACES pain rating scale, TBPI, transcutaneous partial oxygen pressure, total or pain‐free walking distance, and ulcer size serially improved in all patients. No death or major amputation occurred, and severe adverse events were rare, although mild to moderate events relating to G‐CSF and leukapheresis were frequent during the 12‐week follow‐up. In conclusion, the outcomes of this prospective clinical study indicate the safety and feasibility of CD34+ cell therapy in patients with CLI. Favorable trends in efficacy parameters encourage a randomized and controlled trial in the future. STEM CELLS 2009;27:2857–2864

Cardiac Endothelin-1 Plays a Critical Role in the Functional Deterioration of Left Ventricles During the Transition From Compensatory Hypertrophy to Congestive Heart Failure in Salt-Sensitive Hypertensive Rats

BACKGROUND To investigate whether endogenous ET-1 participates in an adaptive process of left ventricular hypertrophy (LVH) or a maladaptive process from LVH to congestive heart failure (CHF), we used a Dahl salt-sensitive (DS) rat model, in which systemic hypertension caused compensated concentric LVH at the age of 11 weeks followed by marked LV dilatation and global hypokinesis at the age of 17 weeks. METHODS AND RESULTS By specific sandwich enzyme immunoassay, serum and myocardial ET-1 levels at the LVH stage were not elevated compared with age-matched Dahl salt-resistant (DR) rats, despite the marked increase of LV/body weight ratio (LV/BW). However, at the CHF stage, serum and LV ET-1 levels increased by 3. 8-fold and 5.4-fold, respectively. LV ET-1 contents had close relationships with the fractional shortening (r=0.763) and the systolic wall stress (r=0.858) measured by in vivo transthoracic echocardiography. Immunohistochemistry demonstrated that the remarkably increased ET-1 in LV is located mainly in cardiomyocytes. By competitive reverse transcriptase-polymerase chain reaction, LV prepro-ET-1 mRNA levels increased by 4.1-fold in CHF rats. We randomized 11-week-old LVH rats to chronic treatment with the endothelin receptor antagonist bosentan (Bos, 100 mg. kg-1. d-1, n=14), the alpha1-receptor antagonist doxazosin (Dox, 1 mg. kg-1. d-1, n=12), or vehicle (Cont, n=14). Bos treatment did not alter the LV geometry and function at 15 weeks; however, it attenuated the decrease of LV fractional shortening by 51% (P<0.01) without reducing the LV/BW at 17 weeks. Conversely, Dox, which decreased the blood pressure to the same extent as Bos, did not affect the progression of LV dysfunction. Bos (93%; P<0.0001 versus Cont) but not Dox (42%; P=0.8465 versus Cont) ameliorated the survival rate at 17 weeks (Cont; 36%). CONCLUSIONS The accelerated myocardial synthesis of ET-1 contributes directly to LV contractile dysfunction during the transition from LVH to CHF. Unelevated levels of LV ET-1 at the established LVH stage and lack of effects on LV mass by chronic bosentan treatment suggest that myocardial growth is mediated through alternative pathways. These studies indicate that chronic ET antagonism may provide an additional strategy for heart failure therapy in humans.

Characterization of Noncalcified Coronary Plaques and Identification of Culprit Lesions in Patients With Acute Coronary Syndrome by 64-Slice Computed Tomography

OBJECTIVES We sought to characterize noncalcified coronary atherosclerotic plaques in culprit and remote coronary atherosclerotic lesions in patients with acute coronary syndrome (ACS) with 64-slice computed tomography (CT). BACKGROUND Lower CT density, positive remodeling, and adjacent spotty coronary calcium are characteristic vessel changes in unstable coronary plaques. METHODS Of 147 consecutive patients who underwent contrast-enhanced 64-slice CT examination for coronary artery visualization, 101 (ACS; n = 21, non-ACS; n = 80) having 228 noncalcified coronary atherosclerotic plaques (NCPs) were studied. Each NCP detected within the vessel wall was evaluated by determining minimum CT density, vascular remodeling index (RI), and morphology of adjacent calcium deposits. RESULTS The CT visualized more NCPs in ACS patients (65 lesions, 3.1 +/- 1.2/patient) than in non-ACS patients (163 lesions, 2.0 +/- 1.1/patient). Minimum CT density (24 +/- 22 vs. 42 +/- 29 Hounsfield units [HU], p < 0.01), RI (1.14 +/- 0.18 vs. 1.08 +/- 0.19, p = 0.02), and frequency of adjacent spotty calcium of NCPs (60% vs. 38%, p < 0.01) were significantly different between ACS and non-ACS patients. Frequency of NCPs with minimum CT density <40 HU, RI >1.05, and adjacent spotty calcium was approximately 2-fold higher in the ACS group than in the non-ACS group (43% vs. 22%, p < 0.01). In the ACS group, only RI was significantly different between 21 culprit and 44 nonculprit lesions (1.26 +/- 0.16 vs. 1.09 +/- 0.17, p < 0.01), and a larger RI (> or = 1.23) was independently related to the culprit lesions (odds ratio: 12.3; 95% confidential interval: 2.9 to 68.7, p < 0.01), but there was a substantial overlap of the distribution of RI values in these 2 groups of lesions. CONCLUSIONS Sixty-four-slice CT angiography demonstrates a higher prevalence of NCPs with vulnerable characteristics in patients with ACS as compared with stable clinical presentation.

Excessive activation of matrix metalloproteinases coincides with left ventricular remodeling during transition from hypertrophy to heart failure in hypertensive rats

OBJECTIVES We sought to elucidate how the local activation of matrix metalloproteinases (MMPs) is balanced by that of the endogenous tissue inhibitors of MMP (TIMPs) during left ventricular (LV) remodeling. BACKGROUND Although it is known that the extracellular matrix (ECM) must be altered during LV remodeling, its local regulation has not been fully elucidated. METHODS In Dahl salt-sensitive rats with hypertension, in which a stage of concentric, compensated left ventricular hypertrophy (LVH) at 11 weeks is followed by a distinct stage of congestive heart failure (CHF) with LV enlargement and dysfunction at 17 weeks, we determined protein and messenger ribonucleic acid (mRNA) levels of LV myocardial TIMP-2 and -4 and MMP-2, as well as their concomitant activities. RESULTS No changes were found at the LVH stage. However, during the transition to CHF, TIMP-2 and -4 activities, protein and mRNA levels were all sharply increased. At the same time, the MMP-2 mRNA and protein levels and activities, as determined by gelatin zymography, as well as by an antibody capture assay, showed a substantial increase during the transition to CHF. The net MMP activities were closely related to increases in LV diameter (r = 0.763) and to systolic wall stress (r = 0.858) in vivo. CONCLUSIONS Both TIMPs and MMP-2 remained inactive during hypertrophy, per se; they were activated during the transition to CHF. At this time, the activation of MMP-2 surpassed that of TIMPs, possibly resulting in ECM breakdown and progression of LV enlargement.

Role of the left atrium in adaptation of the heart to chronic mitral regurgitation in conscious dogs.

The manner in which the left atrium adapts to chronic mitral regurgitation and the role of the adapted left atrium as a modulator of excessive central blood volume were analyzed in seven conscious dogs, instrumented with high-fidelity pressure transducers and ultrasonic dimension gauges for measurement of left atrial and left ventricular pressure and cavity size. After obtaining data in a control situation, mitral regurgitation was produced by transventricular chordal sectioning. Heart rate was matched by right atrial pacing. In the “early” stage (7–14 days), left ventricular end-diastolic and mean left atrial pressures increased from 6 to 16 mm Hg and from 4 to 12 mm Kg, respectively. Both left ventricular end-diastolic segment length and left atrial diameter prior to atrial contraction increased by 7%. In the “late” stage (20–35 days), despite significant decreases in left ventricular filling pressure (11 mm Hg) and left atrial pressure (8 mm Hg), there was a continuous increase in left ventricular end-diastolic dimension (10%) and atrial end-diastolic diameter (10%). After the onset of mitral regurgitation, the left atrium performed greater work with a more enlarged cavity. Left atrial chamber stiffness was progressively decreased. These changes were associated with progressive increase in the left atrial diameter at zero stress, and there was a significant increase in the diameter of the left atrial myocyte. These results indicate that during chronic mitral regurgitation, the left atrium enlarges in size and mass, with a more potent booster action. The left atrial chamber becomes more compliant. Thus, the enlarged left atrium appears to exert an important compensatory mechanism in the case of excessive central blood volume by buffering pressure rise in the atrium and by providing an adequate ventricular filling volume.

Oral infection-inflammatory pathway, periodontitis, is a risk factor for endothelial dysfunction in patients with coronary artery disease

OBJECTIVE Several studies have shown that periodontitis is a risk factor for cardiovascular diseases. There is an association between inflammation and endothelial dysfunction. The purpose of this study was to evaluate endothelial function in patients with coronary artery disease (CAD) who had periodontitis. METHODS AND RESULTS We evaluated forearm blood flow (FBF) responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and to sodium nitroprusside (SNP), an endothelium-independent vasodilator, in 101 CAD patients with periodontitis (37 men and 11 women, 63+/-12 yr) and without periodontitis (36 men and 17 women, 62+/-13 yr). FBF was measured by using strain-gauge plethysmography. Circulating levels of C-reactive protein and interleukin-6 were significantly higher in the periodontitis group than in the non-periodontitis group. FBF response to ACh was significantly smaller in the periodontitis group than in the non-periodontitis group. SNP-stimulated vasodilation was similar in the two groups. Periodontal therapy reduced serum concentrations of C-reactive protein from 2.7+/-1.9 to 1.8+/-0.9mg/L (P<0.05) and interleukin-6 from 2.6+/-3.4 to 1.6+/-2.6ng/L (P<0.05) and augmented ACh-induced vasodilation from 14.7+/-5.2 to 20.1+/-6.1mL/(min100mL) tissue (P<0.05) in patients with periodontitis. The SNP-stimulated vasodilation was similar before and after treatment. After administration of N(G)-monomethyl-l-arginine, a nitric oxide synthase inhibitor, FBF response to ACh was similar before and after treatment. CONCLUSION These findings suggest that periodontitis is associated with endothelial dysfunction in patients with CAD through a decrease in nitric oxide bioavailability. Systemic inflammation may be, at least in part, a cause and predictor of progression of endothelial dysfunction.

Intracellular calcium and ventricular fibrillation. Studies in the aequorin-loaded isovolumic ferret heart.

To elucidate the role of changes in [Ca2+]i in the induction of ventricular fibrillation (VF), Ca2+i signals, epicardial electrical potentials, and isovolumic left ventricular pressure were simultaneously recorded in isolated intact ferret hearts loaded with aequorin, a bioluminescent protein. When the preparations were perfused with 3 microM acetylstrophanthidin and 8 mM Ca2+, or with a low Na+ solution (18 mM Na+, 100 mM Li+), spontaneous transitions to the VF state were consistently observed within a short period of time. The initiation of spontaneous VF was preceded by development of a Ca2+i overload state, coincidental with the ascending phase of diastolic Ca2+i oscillations, and was followed by further elevation in Ca2+i levels, which were associated with augmented Ca2+i oscillations of a saw-toothed pattern. Pretreatment with 10 microM ryanodine, which blocked Ca2+i oscillations in the preparation, did not eliminate inducibility of VF by means of AC electrical stimulations; however, VF no longer occurred spontaneously, and the threshold for VF induction increased markedly. In the absence of a state of Ca2+i overload, spontaneous defibrillation occurred within a minute after the initiation of VF. We conclude that 1) VF can be induced in the absence of Ca2+i oscillations; however, 2) Ca2+i oscillations play a crucial role as a trigger for VF and therefore are an important determinant of the vulnerability to VF; and 3) the augmented Ca2+i oscillations after the transition to VF state may support the maintenance of this type of arrhythmia.

Anti-Ischemic Effect of a Novel Cardioprotective Agent, JTV519, Is Mediated Through Specific Activation of δ-Isoform of Protein Kinase C in Rat Ventricular Myocardium

BACKGROUND A new 1,4-benzothiazepine derivative, JTV519, has a strong protective effect against Ca(2+) overload-induced myocardial injury. We investigated the effect of JTV519 on ischemia/reperfusion injury in isolated rat hearts. METHODS AND RESULTS At 30 minutes of reperfusion after 30-minute global ischemia, the percent recovery of left ventricular developed pressure was improved, and the creatine phosphokinase and lactate dehydrogenase leakage was reduced in a concentration-dependent manner when JTV519 was administered in the coronary perfusate both at 5 minutes before the induction of ischemia and at the time of reperfusion. The myocardial protective effect of JTV519 was completely blocked by pretreatment of the heart with GF109203X, a specific protein kinase C (PKC) inhibitor. In contrast, the effect of JTV519 was not affected by alpha(1)-, A(1)-, and B(2)-receptor blockers that couple with PKC in the cardiomyocyte. Both immunofluorescence images and immunoblots of JTV519-treated left ventricular myocardium and isolated ventricular myocytes demonstrated that this agent induced concentration-dependent translocation of the delta-isoform but not the other isoforms of PKC to the plasma membrane. CONCLUSIONS The mechanism of cardioprotection by JTV519 against ischemia/reperfusion injury involves isozyme-specific PKC activation through a receptor-independent mechanism. This agent may provide a novel pharmacological approach for the treatment of patients with acute coronary diseases via a subcellular mechanism mimicking ischemic preconditioning.