Ronald W. Millard

Regional myocardial functional and electrophysiological alterations after brief coronary artery occlusion in conscious dogs.

The time relationship for recovery of mechanical function, the intramyocardial electrogram and coronary flow after brief periods of regional myocardial ischemia, was studied in conscious dogs. Total left vemtricular (LV) function was assessed with measurements of LV systolic and diastolic pressures, rate of change of LV pressure (dP/dt), and dP/dt/P. Regional LV function was assessed with measurements of regional segment length and velocity of shortening. An implanted hydraulic occluder on either the left anterior descending or circumflex coronary artery was inflated for 5- and 15-min periods on separate days. A 5-min occlusion depressed overall LV function transiently, but just before release of occlusion overall function had nearly returned to control. At this time regional function in the ischemic zone was still depressed to the point of absent shorteining or paradoxical motion during systole and was associated with marked ST segment elevation (+ 10 +/- 2.2 mV) at the site where function was measured. With release of occlusion and reperfusion the intramyocardial electrogram returned to normal within 1 min, and reactive hyperemia subsided by 5-10 min. In contrast to the rapid return to preocclusion levels for coronary flow and the electrogram, regional mechanical function remained depressed for over 3 h. A 15-min coronary occlusion resulted in an even more prolonged (greater than 6 h) derangement of function in the ischemic zone. Thus, brief periods of coronary occlusion result in prolonged impairement of regional myocardial function which could not have been predicted from the rapid return of the electrogram and coronary flow. These observations indicate that brief interruptions of coronary flow result either in a prolonged period of local ischemia or that alterations of mechanical induced by ischemia far outlast the repayment of the oxygen debt.

Effects of isoproterenol on regional myocardial function, electrogram, and blood flow in conscious dogs with myocardial ischemia.

The effects of coronary occlusion and of subsequent isoproterenol infusion were examined in conscious dogs. Left ventricular (LV) function was assessed by measurements of LV diameter, pressure, velocity and dP/dt/P, and regional myocardial function was assessed by measurements of segment length (SL) and velocity of SL shortening in normal, border, and ischemic zones. Regional myocardial function was measured from the same sites, along with intramyocardial electrograms and regional myocardial blood flow as determined by radioactive microspheres. Coronary occlusion resulted in graded loss of function from the normal to severely ischemic zones with graded flow reductions and graded increases in ST segment elevation. Isoproterenol improved overall LV function, and function in the normal zone. Isoproterenol also improved function in 19 of 21 border-zone segments and in all moderately ischemic segments, while elevating further the ST segments. These changes were accompanied by increases in myocardial blood flow. In contrast, in severely ischemic segments, isoproterenol resulted in a deterioration of function, in that paradoxical motion occurred in segments previously akinetic during systole, while paradoxical motion was intensified in those segments in which it was already present. These changes were accompanied by further ST segment elevation but not by concurrent increases in blood flow. In addition, in 2 of 21 border zone segments, myocardial blood flow fell and these segments responded to isoproterenol with complete loss of function; paradoxical motion developed. Thus, in the conscious dog, a strong inotropic agent can improve function, even in the ischemic myocardium, as long as the required additional blood flow can be provided wither through primary or collateral channels.

Cardiomyocytes mediate anti-angiogenesis in type 2 diabetic rats through the exosomal transfer of miR-320 into endothelial cells

Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play critical roles in mediating cell-to-cell communication. Given that diabetic hearts exhibit insufficient angiogenesis, it is significant to test whether diabetic cardiomyocyte-derived exosomes possess any capacity in regulating angiogenesis. In this study, we first observed that both proliferation and migration of mouse cardiac endothelial cells (MCECs) were inhibited when co-cultured with cardiomyocytes isolated from adult Goto-Kakizaki (GK) rats, a commonly used animal model of type 2 diabetes. However, GK-myocyte-mediated anti-angiogenic effects were negated upon addition of GW4869, an inhibitor of exosome formation/release, into the co-cultures. Next, exosomes were purified from the myocyte culture supernatants by differential centrifugation. While exosomes derived from GK myocytes (GK-exosomes) displayed similar size and molecular markers (CD63 and CD81) to those originated from the control Wistar rat myocytes (WT-exosomes), their regulatory role in angiogenesis is opposite. We observed that the MCEC proliferation, migration and tube-like formation were inhibited by GK-exosomes, but were promoted by WT-exosomes. Mechanistically, we found that GK-exosomes encapsulated higher levels of miR-320 and lower levels of miR-126 compared to WT-exosomes. Furthermore, GK-exosomes were effectively taken up by MCECs and delivered miR-320. In addition, transportation of miR-320 from myocytes to MCECs could be blocked by GW4869. Importantly, the exosomal miR-320 functionally down-regulated its target genes (IGF-1, Hsp20 and Ets2) in recipient MCECs, and overexpression of miR-320 inhibited MCEC migration and tube formation. GK exosome-mediated inhibitory effects on angiogenesis were removed by knockdown of miR-320. Together, these data indicate that cardiomyocytes exert an anti-angiogenic function in type 2 diabetic rats through exosomal transfer of miR-320 into endothelial cells. Thus, our study provides a novel mechanism underlying diabetes mellitus-induced myocardial vascular deficiency which may be caused by secretion of anti-angiogenic exosomes from cardiomyocyes.

Reflex chronotropic and inotropic effects of calcium channel-blocking agents in conscious dogs. Diltiazem, verapamil, and nifedipine compared.

In chronically instrumented, conscious dogs, rapid injection of equihypotensive doses of three calcium channel-blocking agents, verapamil (250 μg/kg), diltiazem (200 μg/kg) and nifedi-pine (50 μg/kg), produced disparate chronotropic and inotropic responses. Although they all decreased mean arterial pressure by about 10%, heart rate (93 ± 4 beats/min) was markedly increased to 175 ± 12 with nifedipine, to 163 ± 15 with verapamil, and only slightly increased to 118 ± 7 with diltiazem. Contractile responses measured before (left ventricular dP/dtmax, 2749 ± 131 mm Hg/sec) and during left ventricular ejection (endocardial dimension dD/dtmax, 57 ± 4 mm/sec) were increased by 24% and 14% with nifedipine, decreased by 26% and 22% with verapamil, and were unchanged with diltiazem. These chronotropic and inotropic responses to rapid intravenous administration of the three drugs were increased in a dose-dependent manner. Similar results also were observed after slow infusion of these drugs. To determine the extent to which autonomic reflexes participated in these cardiac responses, propranolol (0.5 mg/kg) or propranolol plus atropine (0.1–0.2 mg/kg) was administered prior to injection of each calcium channel-blocking agent. Propranolol abolished the positive inotropic response to nifedipine and potentiated the negative inotropic response to verapamil. Positive chronotropic responses to verapamil, nifedipine, and diltiazem were attenuated by propranolol plus atropine. These results suggest that equihypotensive doses of the three prominent calcium channel-blocking agents exert different degrees of autonomic reflex activation in awake, unsedated dogs. These reflexes, which modulate the direct effects of calcium channel-blocking agents on chronotropic and inotropic variables of the heart, may have important clinical implications.

Exosomes secreted from GATA-4 overexpressing mesenchymal stem cells serve as a reservoir of anti-apoptotic microRNAs for cardioprotection.

BACKGROUND Exosomes play an important role in intercellular signaling and exert regulatory function by carrying bioactive molecules. This study investigated (1) the cardioprotective capabilities of exosomes derived from mesenchymal stem cells (MSCs) overexpressing GATA-4 (MSC(GATA-4)) and (2) its underlying regulatory mechanisms for expression of target proteins in recipient cells. METHODS AND RESULTS Exosomes were isolated and purified from MSC(GATA-4) (Exo(GATA-4)) and control MSCs (Exo(Null)). Cell injury was investigated in primary cultured rat neonatal cardiomyocytes (CM) and in the rat heart. Exosomes contributed to increased CM survival, reduced CM apoptosis, and preserved mitochondrial membrane potential in CM cultured under a hypoxic environment. Direct intramyocardial transplantation of exosomes at the border of an ischemic region following ligation of the left anterior descending coronary artery significantly restored cardiac contractile function and reduced infarct size. Real-time PCR revealed that several anti-apoptotic miRs were highly expressed in Exo(GATA-4). Rapid internalization of Exo(GATA-4) by CM was documented using time-lapse imaging. Subsequent expression of these miRs, particularly miR-19a was higher in CM and in the myocardium treated with Exo(GATA-4) compared to those treated with Exo(Null). The enhanced protective effects observed in CM were diminished by the inhibition of miR-19a. The expression level of PTEN, a predicted target of miR-19a, was reduced in CM treated with Exo(GATA-4), which resulted in the activation of the Akt and ERK signaling pathways. CONCLUSIONS Exo(GATA-4) upon transplantation in the damaged tissue mediate protection by releasing multiple miRs responsible for activation of the cell survival signaling pathway.

Inadequate cardioplegic protection with obstructed coronary arteries.

Abstract To determine the contribution of complete cardioplegia to the preservation of left ventricular (LV) function, we put ultrasonic transducers in the anterior and posterior walls of the left ventricle in 18 dog hearts. The dogs were subjected to global ischemia for 60 minutes at 28°C, and the speed of segment shortening (dl/dt) and percent of systolic shortening of the two wall regions before and after ischemic manipulations were measured. When cardioplegic perfusion was uniform, there was no significant difference between the anterior and posterior walls in any of the variables measured, and global LV function (stroke work) was well preserved. However, when the left anterior descending coronary artery was occluded during cardioplegic infusion, there was significant dysfunction after reperfusion of the anterior wall: without perfusion, the anterior segments recovered only 41% (5.9/14.3 mm/sec) of preischemic dl/dt, while the perfused anterior segments retained 78% (11.4/14.6 mm/sec) of control dl/dt ( p p Regionally inadequate cardioplegic protection during coronary artery bypass graft operation may contribute to perioperative infarction and LV dysfunction, and appropriate timing of anastomoses to ensure early cardioplegic perfusion of all ischemic myocardium is important.

Connective tissue changes in early ischemia of porcine myocardium: An ultrastructural study

Scanning electron microscopy and transmission electron microscopy were used together with tannic acid and ruthenium-red staining to examine connective tissue damage caused by acute myocardial ischemia for 20, 40 and 120 min in pig hearts. The microsphere blood flow technique revealed that blood flow was approximately 0.02 ml/min/g in inner, middle and outer thirds of the ischemic zone. After 20 min of occlusion of the left anterior descending coronary artery, the collagen network and microfilaments became irregularly arranged. After 40 min of occlusion, ruthenium-red positive glycoprotein material around the collagen fibrils and elastin began to disappear. After 2 h occlusion, the collagen fibrils and microfilaments had separated from the basement membrane. Collagen fibrils, elastic fibers, and microfilaments were broken down and were found in decreased quantities. These results have revealed that the connective tissue remains intact during the first 20 min of coronary occlusion despite zero blood flow and mild cellular changes but does undergo prominent alterations after 40 min of occlusion.

Transmural cellular damage and blood flow distribution in early ischemia in pig hearts.

Transmural histological changes were determined morphometrically in the left ventricular free wall of 20 pigs after Iigation of distal left anterior descending coronary artery for 10, 20, 40, and 120 minutes. Hemodynamics were recorded and regional blood flow distribution was measured in the ischemic zone. Coronary occlusion produced regional transmural ischemia without producing significant systemic hemodynamic change. The microsphere blood flow technique revealed that blood flow was less than 0.05 ml/min per g in all layers of the ischemic zone, i.e., inner, middle, and outer thirds. Ischemic cellular damage was classified and quantified from grade O to grade 5 (0 being normal and 5 being the most severe damage) with light microscopy and confirmed by electron microscopy. Layers of 200 μm immediately beneath the endocardium and epicardium showed minimal ischemic damage of less than grade 1.4 regardless of duration of ischemia in all hearts. In the ischemic left ventricular wall, except for the above layers, a definite transmural gradient of the cellular damage existed from the inner third (grade 2.3 ± 0.1) to the outer third (grade 1.3 ± 0.2) at 20 minutes of ischemia and at 40 minutes of ischemia (grades 3.6 ± 0.1 and 1.9 ± 0.3, respectively). The transmural ischemic damage gradient disappeared at 120 minutes of ischemia, where the inner and outer third ischemic grades were both 5.0 ± 0.1. The data suggest that the limited ischemic damage which occurs in the few cell layers beneath endocardium and epicardium may be explained by regional collateral blood flow. An early ischemic damage wavefront phenomenon does exist in the pig myocardium and is independent of myocardial blood flow and its distribution. The transmural cell damage gradient may be the result of transmural gradients of wall stress and intramyocardial pressure in vivo. Therefore, it appears that factors other than blood flow are the major determinants of ischemic cellular damage in the left ventricular wall of hearts lacking a collateral blood supply.

Effects of diltiazem, a calcium antagonist, on regional myocardial function and mitochondria after brief coronary occlusion

Abstract The purpose of the study was to assess the effect of the calcium antagonist diltiazem on mechanical and mitochondrial function of ischemic myocardium of the dog. Persistent depression of developed tension following brief coronary occlusion was measured in anesthetized and thoracotomized dogs. The extent of persistent depression of developed tension during reperfusion depended on the duration of occlusion and also on the pressure-rate index during occlusion. Diltiazem prevented the marked drop in developed tension of the ischemic segment of the myocardium following 5 min or 10 min of coronary occlusion. The inactive optical isomer of diltiazem had no effect on developed tension before or after coronary occlusion. Depression of the state 3 rate of respiration of mitochondria observed following 10 min of occlusion of coronary artery was almost completely reversed by pretreatment with diltiazem. Diltiazem may reduce the damage of ischemic myocardium during occlusion by hemodynamic action of the drug, and possibly by preventing damage to mitochondria.

Aortic blood flow and cardiac output in the hemoglobin-free fish Chaenocephalus aceratus.

Abstract 1. 1. The cardiac output of Chaenocephalus aceratus, determined by the Fick principle, ranged from 99 to 153 ml/kg per min in unrestrained specimens at rest. These values are several-fold higher than those of other fishes. 2. 2. The combined energy cost for cardiac and respiratory work at rest was estimated to be nearly half, or more, of the total oxygen consumption. 3. 3. The ventral aortic blood flow, measured by electromagnetic flowmeter, increased during hypoxia and decreased during hyperoxia. The cardiac output was regulated by changes in the stroke volume and not heart rate. 4. 4. The ventral and/or dorsal aortic blood pressures were monitored simultaneously with the blood flow. 5. 5. Infusions of cardio- and vasoactive drugs revealed typical responses.