The effect of verapamil on myocardial ultrastructure during and following release of coronary artery occlusion

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

36, 277-286 (1982)

The Effect of Verapamil on Myocardial and following Release of Coronary

Ultrastructure during Artery Occlusion1

ROBERTA. KLONER,~ LAURENCE W. V. DEBOER, NANCY CARLSON, AND EUGENE BRAUNWALD Department

of Medicine,

Received

Brigham and Women’s Hospital and Harvard Boston, Massachusetts 02115 June

30, 1981,

and in revised

form

August

Medical

School,

21, 1981

Although the calcium channel blocking agent verapamil has been shown to have beneficial effects on ischemic myocardium, its effect on cardiac ultrastructure during regional myocardial ischemia and following coronary reperfusion has not been studied in detail. The purpose of this study was to investigate the effect of verapamil on the ultrastructure of myocardium during the early phase of ischemia and following coronary reperfusion. Open-chest anesthetized dogs were subjected to 1 hr of occlusion of the proximal left anterior descending coronary artery followed by 1 hr of reperfusion. The mean ischemic score, a semiquantitative index of ultrastructural damage, was significantly lower in verapamil-treated (0.9 ? 0.3) than in untreated dogs (1.9 ? 0.2, P < 0.025) during coronary occlusion and especially following reperfusion (1.0 -C 0.3 versus 2.9 2 0.5, respectively P < 0.025). Verapamil treatment prevented the hastening of ultrastructural damage (explosive cell swelling phenomenon) associated with reperfusion into severely ischemic myocardium. Verapamil resulted in a more profound reduction of the extent of ultrastructural damage of mitochondria compared to other organelles. Thus, this study supports the concepts that verapamil reduces ultrastructural damage both during coronary occlusion and following coronary reperfusion and may reduce ischemic damage by protecting mitochondrial structure.

INTRODUCTION Although several pharmacologic agents have been shown to decrease the size of experimental myocardial infarction (Kloner et al., 1978a, 198Oa), the mechanism of their beneficial effects is not clear. Study of how pharmacologic agents affect the cardiac ultrastructure in acute ischemia may offer insight into their mode of action (Kloner et al., 1978b). Electron microscopic techniques permit determination of whether an intervention alters the early morphologic sequence of events after coronary occlusion and whether it protects some organelles preferentially. We recently reported that the calcium blocking agent, verapamil, reduces myocardial infarct size after experimental coronary artery occlusion (DeBoer et al., 1980). With recent interest in treating acute myocardial infarction with reperfusion it would be useful to assess whether a pharmacologic agent can protect severely ischemic subendocardial myocardium both during the period of &hernia and following reperfusion. The purpose of this study was to determine whether verapamil could prevent ultrastructural cellular damage during ischemia and prevent the hastening of ultrastructural damage in severely ischemic subendocardium following coronary reperfusion. Grading systems were employed in order to determine whether there was a disproportionate preservation of any of the organelles. * Supported in part by Grants HL-23140 and SCOR 26215 under the National Heart, Lung and Blood Institute, Bethesda, Md. 2 Established Investigator of the American Heart Association. To whom reprint requests should be sent at: Harvard Medical School, 180 Longwood Ave., Room 235, Boston, Mass. 02115. 277 0014-4800/82/030277-10$02.00/O Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.

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METHODS Thirteen mongrel dogs were anesthetized with 20 mg/kg of intravenous sodium thiamylal. They were intubated and ventilated with room air using a Harvard respirator. The left jugular vein and left common carotid artery were cannulated. Arterial pressure was monitored with a Statham P23Db pressure transducer and electrocardiographic lead aVF was monitored and recorded on a Brush multichannel recorder (Gould Co., Cleveland, Ohio). After performing a left thoracotomy in the fifth intercostal space, the heart was suspended in a pericardial cradle. The proximal left anterior descending coronary artery was dissected free and occluded with a Schwartz vascular clamp at a site proximal to the first large diagonal branch. All hearts developed anteroapical cyanosis which was apparent visually. Regional myocardial blood flow was analyzed 3 min after coronary artery occlusion with radioactive plastic microspheres (9 ? 1 pm labeled with “?Sn or 46Sc) as previously described (Kloner et al., 1978a). Approximately 2 million spheres were injected for each determination. The coronary occlusion was maintained for 1 hr, the clamp was then removed, and coronary reperfusion was carried out for 1 hr. In vivo biopsies for ultrastructural analysis from the central ischemic zone, as defined by the distribution of epicardial cyanosis and distribution of the occluded left anterior descending vessel were obtained with a specially designed 8-gauge needle biopsy tool. Three in vivo biopsies from the central ischemic zone and three from the remote nonischemic zone were obtained 1 hr after coronary occlusion (just prior to reperfusion) and after 1 hr of coronary reperfusion. The subendocardial half of the sample was placed into Karnovsky’s fixative (2.5% glutaraldehyde and 2.0% paraformaldehyde cacodylate buffer), pH 7.40, for 3 hr. The subendocardium was chosen for analysis since this is the zone of tissue which first shows ischemic damage following 1 hr of coronary occlusion (Jennings et al., 1975; Whalen et al., 1974). The samples were washed in cacodylate buffer and postfixed with 1% osmic acid for 1 hr, dehydrated with alcohol, and embedded in Epon 812. Thin sections were cut on a Porter-Blum microtome, mounted on plain copper grids, stained with aqueous uranyl acetate and lead citrate, and examined on a Philips electron microscope. Dogs were randomized to saline (n = 7)- or verapamil (n = 6)-treated groups (0.2 mg/kg intravenously 5 min after coronary occlusion followed by a continuous infusion 0.6 mg/kg/hr). Saline or verapamil was continued until 1 hr after coronary reperfusion at which time the dogs were sacrificed by an overdose of barbiturate. Morphometric analysis of electron micrographs. For detailed analysis of organelle changes in the myocardial cells, a mean ischemic score (Kloner et al., 1978b, 1980b), a semiquantitative assessment of myocardial cell injury, was calculated on approximately 200 electron micrographs (magnification x 17,500 to 37,000). To verify whether verapamil preferentially protects the ultrastructure of mitochondria, overall myocardial cell injury was ranked independently of mitochondrial structure from 0 to 4 as follows: 0 = normal (other than mitochondrial change). 1 = minimal ischemic changes (nuclear chromatin clumping and margination. I bands, glycogen loss). 2 = moderate ischemic changes (the findings in 1, plus early intermyotibrillar and sarcoplasmic reticular edema). 3 = severe ischemic changes (findings in 2, plus subsarcolemmal blebs, sarcolemmal gaps, and marked edema).

EFFECT

OF

VERAPAMIL

4 = total architectural disruption sarcolemmal membrane).

ON

ISCHEMIC

MYOCARDIUM

279

(with loss of sarcomere structure and absent

The severity of mitochondrial swelling was based upon a previously grading system (Kloner et al., 1978b).

described

0 = normal mitochondria. 1 = early swelling as manifested by clearing of matrix density and separation of cristae. 2 = more marked swelling as manifested by further clearing of matrix density and separation of cristae. 3 = massive swelling with disruption of cristae. 4 = massive swelling with disruption of cristae and rnpture of inner and outer mitochondrial membranes. Mitochondria were assigned a numerical value of 0 to 4, depending on its degree of morphologic damage. A mean mitochondrial grade for each cell was obtained by calculating the weighted average (0 to 4) of mitochondrial swelling. The number of intramitochondrial dense bodies was counted. Approximately 3200 mitochondria were graded in this manner. The degree of sarcoplasmic reticular swelling was graded 0 to 3 (0 = absent, 3 = severely altered, 1 and 2 = intermediate); the presence of glycogen and lipid was ranked as 0 to 3 (0 = absent, 1 = sparse, 2 = moderately abundant, and 3 = more abundant). The presence or absence of large contraction bands was recorded. At the time of grading the examiners did not know whether electron micrographs were from untreated or treated groups. Regional myocardial blood jlow analysis. Following excision of the hearts, biopsy sites for electron microscopy were identified and 0.7 to 0.8-g samples of subendocardial myocardium were obtained from tissue contiguous to the biopsy sites in both nonischemic and ischemic zones. Tissue and reference sample blood were counted in a gamma counter. Regional myocardial blood flow (RMBF) analysis was calculated as previously described (Kloner et al., 1978a). RESULTS Qualitative

Assessment

One dog in the verapamil group died of ventricular fibrillation and was excluded from the study. Myocardium from the nonischemic subendocardial region appeared normal, at 1 hr after coronary occlusion and following 1 hr of coronary reperfusion, both in saline- and verapamil-treated dogs. After 1 hr of coronary occlusion, myocardium from the central ischemic region in saline-treated control dogs showed clumping and margination of nuclear chromatin, wide I bands, loss of glycogen, swollen mitochondria with numerous amorphous dense bodies, vacuoles, intermyofibrillar edema, and sarcolemmal breaks (Fig. 1A). After 1 hr of coronary reperfusion the cell swelling was more marked with lifting of the sarcolemmal membrane off the body of the myofilaments, contraction bands, increased vacuolization, and granular dense bodies in the mitochondria (Fig. 1B). This phenomenon, termed “explosive cell swelling,” has previously been described in severely ischemic subendocardial tissue subjected to coronary reperfusion (Jennings et al., 1975) and reflects the fact that irreversibly injured tissue has lost its ability to control cell volume (Whalen et al., 1974).

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FIG. 1A. Untreated ischemic myocardium following 1 hr of coronary occlusion. The cell is swollen as manifested by intracellular edema(e): I bands (I) are present and glycogen is absent. The nucleus (n) shows chromatin clumping and margination. The mitochondria (m) are swollen with loss of matrix density, separation of cristae, and amorphous dense bodies (arrows) (X 11,000).

FIG. 1B. Untreated myocardium following 1 hr of coronary occlusion plus 1 hr of reperfusion. The cell is markedly swollen with disruption of cellular architecture and large contraction bands (cb). 1‘here is separation of mitochondrial cristae and numerous dense bodies within the mitochondria (arr ows) (X11,000).

EFFECT

OF VERAPAMIL

ON ISCHEMIC

MYOCARDIUM

281

FIG. IC. Verapamil-treated myocardium after 1 hr of &hernia. The degree of overall cellular damage : is less than that in (A). Intermyotibrillar edema is present and glycogen granules arce sparse. Mitochl ondria (m) appear intact (X 11,000).

FIG. ID. Verapamil-treated myocardium following 1 hr of ischemia plus 1 hr of coronary reperfusion. T‘he overall cellular architecture is intact. Sarcomeres are somewhat contracted but no large contrac :tion bands are present. The nucleus (n) appears normal. There is minimal intermy edema. Mitochondria (m) are intact (x 11,000).

KLONER

282

ET AL.

FIG. 2. Veraparnil-treated myocardium subjected to 1 hr of ischemia plus 1 hr of coronary reperfusion in a cell showing marked cellular swelling. Large spaces between myotibrils represent edema (e). Glycogen is sparse and myofilaments show areas of loss of normal architecture (arrows). Despite these changes, the mitochondria appear intact. Compare to Figs 1A and B (X 14,000).

In the verapamil-treated group, fewer cells were damaged, both during ischemia and following reperfusion (Figs. lC, D). Cells that did appear damaged differed from untreated cells in that the mitochondria did not appear swollen and contained fewer dense bodies (Fig. 2). Otherwise, glycogen loss, nuclear change, sarcoplasmic reticular swelling, I bands, intermyofibrillar edema were similar in those cells which appeared damaged to the untreated group. Quantitative

Assessment

While 93 t 5% of the cells in the saline-treated group were damaged (cell grade greater than 0) during the period of coronary occlusion, only 57 + 12% in the verapamil-treated group showed damage at this time period (Z’ < 0.05). Similarly, following reperfusion, while 82 ‘-+ 10% of cells in the untreated group showed myocardial cell damage only 54 2 16% in the verapamil-treated group showed evidence of any damage (P < 0.05). The mean ischemic score (MIS) was signiticantly less in the verapamil-treated dogs compared to the saline-treated controls both during coronary occlusion (0.9 + 0.3 versus 1.9 k 0.2 in verapamil versus untreated, respectively; P < 0.025) and following coronary reperfusion (1 .O +- 0.3 versus 2.9 + 0.5; P < 0.025) (Table I). Thus, there was a significant increase in the MIS following reperfusion in the control dogs (from 1.9 k 0.2 to 2.9 + 0.5, P < 0.05) presumably a manifestation of the explosive cell swelling phenomenon; however, there was no rise in MIS following reperfusion in the verapamil-treated dogs (0.9 k 0.3 to 1.0 k 0.3, NS). Table II shows that the mean grade of mitochondrial injury for any given cellular grade of injury was distinctly lower in the verapamil-treated animals. In this study, no cell grades of 4 were present in the treated group. Mitochondria in Grade

EFFECT

OF VERAPAMIL

Morphometric

ON ISCHEMIC

MYOCARDIUM

TABLE I Analysis of Ultrastructural

Cell mean ischemic score

Control Verapamil

283

Changes Mitochondrial

grade

During occlusion

During reperfusion

During occlusion

During reperfusion

1.9 2 0.2 0.9 + 0.3**

2.9 2 OS* 1.0 ? 0.3**

1.8 k 0.2 0.1 2 0.1***

1.8 ” 0.5 0.1 2 0.1**

* P < 0.05 versus during occlusion. **P < 0.025 versus control. ***P < 0.001 versus control.

0 cells (normal cells) appeared normal in both untreated and treated groups. While the average number of intramitochondrial dense bodies per mitochondria in untreated dogs was 1.30 f .07 following occlusion, it rose to 1.76 + .ll (P < 0.005) following reperfusion; in verapamil-treated animals the number of dense bodies per mitochondria approached zero both during and after occlusion. Large contraction bands were not present in the biopsies obtained during coronary occlusion. However, four of seven animals in the untreated group demonstrated numerous large contraction bands in the ischemically injured zone following coronary reperfusion. Contraction bands were not present in any of the verapamil-treated dogs, either during coronary occlusion or following its release. In those cells showing evidence of ischemic damage, sarcoplasmic reticular edema was similar in untreated and treated animals during occlusion (score of 1.0 2 0.2 versus 0.9 + 0.3 in untreated and verapamil-treated animals, respectively); there was no change in grade following reperfusion. Glycogen was sparse (Grade l), as were lipid droplets (Grade 1) in untreated and treated dogs both during and following reperfusion. Regional Myocardial

Blood Flow Analysis

Regional myocardial blood flow in the remote nonischemic subendocardium analyzed 3 min after coronary artery occlusion but before therapy was 1 .Ol + 0.19

Mitochondrial

TABLE II Changes in Ischemic Myocytes Mean mitochondrial

Cell Grade 0 Untreated Occlusion Reflow Verapamil Occlusion Reflow

-

Cell Grade 1

Cell Grade 2

0

1.7 k 0.4 0

1.7 f 0.1 1.3 * 0.3

0 0

0.3 + 0.1* 0

0* 0.1 k 0.1**

a -, Cell grade not present. * P < 0.001 vs untreated. ** P < 0.005 vs untreated. ***P < 0.05 vs untreated.

grade Cell Grade 3

Cell Grade 4

1.8 2 0.2 1.8 ” 0.6

2.1 f 0.4 2.6 f 0.1

0* o***

-a -

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ml/mm/g in untreated dogs and 1.30 t .10 ml/mm/g of tissue in verapamil-treated dogs (P = NS). Within the ischemic zone, regional subendocardial blood flow was 0.10 ? 0.06 ml/mm/g in untreated dogs and 0.10 & 0.02 ml/mm/g in verapamiltreated dogs prior to therapy. Therefore, the effects of verapamil were not due to any intrinsic differences in collateral flow before therapy. DISCUSSION The results of this study suggest that when administered early following coronary occlusion, verapamil reduces the degree of ultrastructural damage during coronary occlusion and following reperfusion. Furthermore, the drug appears to protect mitochondrial structure even in those cells which appeared otherwise damaged. Verapamil also prevented the development of large contraction bands following reperfusion. Previous studies have suggested that verapamil exerts beneficial effects when administered during myocardial ischemia. It has been shown to reduce myocardial infarct size as assessed by pathologic examination (DeBoer et al., 1980; Reimer et al., 1977a), epicardial ST segment elevation (Smith et al., 1975; Smith et al., 1977; daLuz et al., 1980), and myocardial CK activity (Lefer et al., 1979). In addition, verapamil has been shown to reduce regional coronary vascular resistance (daLuz et al., 1980), and improve the respiratory function of mitochondria extracted from hypoxic heart muscle (Nayler et al., 1978; Clements et al., 1978). Verapamil also exerts beneficial effects upon regional mechanical performance of ischemic myocardium (Sherman et al., 1981). The mechanism of action by which verapamil reduces ischemic damage is not entirely clear but several possibilities have been suggested (Sherman et al., 1981) including (1) a reduction of transmembrane calcium flux into ischemic myocardium; (2) a reduction in afterload and hence, myocardial oxygen consumption; (3) preservation of mitochondrial function; and (4) an improvement in coronary collateral flow to the ischemic region. The present study reveals that verapamil protects the ultrastructure of ischemic cells in a regional model of ischemia both during the coronary occlusion and following reperfusion. In addition, we observed that verapamil produced a disproportionate reduction in damage to mitochondria compared to other organelles. That is, verapamil reduced the percentage of cells in the ischemic zone which appeared damaged by ischemia and the severity of ultrastructural damage in those cells which were damaged. Furthermore, even in those cells which, despite verapamil treatment, did show marked cell swelling, loss of glycogen, and nuclear chromatin clumping, the mitochondria often appeared entirely normal. Thus, our results provide morphologic data which support the hypothesis that verapamil exerts a potent protective effect on mitochondria. They are consistent with the observations of Nayler et al. (1976, 1978), who reported that rabbit hearts perfused by the Langendorff technique and treated with verapamil maintained near normal mitochondrial oxidative phosphorylation and Ca2+ accumulating activities after 60 min of hypoxic perfusion. Similarly, Clements et al. (1978) reported that verapamil improved State 3 respiration of mitochondria which had been made ischemic. We observed that in contrast to the regular appearance of intramitochondrial dense bodies in control dogs during 1 hr of occlusion and an increase in their number during reperfusion, these structures were essentially absent both after ischemia and reperfusion in the verapamil treated animals. A similar protection of mitochondrial ultrastructural damage was

EFFECT

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ON

ISCHEMIC

MYOCARDIUM

285

observed in rats with coronary occlusions treated with propranolol (Kloner et al., 1978b). On the other hand, hyaluronidase-a drug which has also been shown to reduce myocardial infarct size (Kloner et al., 1978a)-did not have this effect on mitochondria (Kloner et al., 1980b). Verapamil also reduced the extent of contraction band formation following reperfusion which may be related to maintenance of ATP stores (Nayler, 1978) possibly due to preserved mitochondrial function. The present study investigated ultrastructural abnormalities in the severely ischemic subendocardium where coronary blood flow is reduced to less than 15% of normal (Becker et al., 1973; Kloner et al., 1976). It is this region of tissue in which irreversible damage first develops. When this tissue is subjected to coronary reperfusion the irreversibly injured cells cannot maintain their volume when faced with the sudden influx of fluid and electrolytes and hence-rapidly accumulate water, Na+, and Ca’+, and show ultrastructural evidence of severe intracellular edema. Jennings et al., refer to this alteration in cells as the “explosive cell swelling phenomenon” (Jennings et al., 1975). This phenomenon should not be interpreted to mean that reperfusion causes increased damage, since cells developing explosive cell swelling are irreversibly injured even before reperfusion (as shown in Fig. 1A). Most studies have shown that reperfusion within 6 hr of coronary occlusion usually salvages subepicardial tissue. Subepicardial tissue remains reversibly injured for a longer period of time than subendocardial tissue (Reimer et al., 1977b) probably due to better coronary collateral flow in this region as well as hemodynamic factors. Reperfusion of reversibly injured subepicardium does not undergo explosive cell swelling. In this study, verapamil protected subendocardial cells during the coronary occlusion with a subsequent reduction in cells which showed explosive cell swelling following reperfusion. In contrast, control animals showed explosive cell swelling following reperfusion with an actual increase in MIS during the reperfusion period. It is likely that the reduction of this cell swelling with verapamil was due to an effect of verapamil on the cells during the period of ischemia since administration of the drug only during reperfusion does not reduce necrosis (Reimer et al., 1977). With recent interest in treatment of acute coronary occlusion by reperfusion techniques (Cohn et al., 1972; Ganz et al., 1981; Phillips et al., 1979; Rentrop et al., 1981) an agent which delays or prevents myocardial cell damage during the critical hours prior to reperfusion may act as a potentially useful holding maneuver. By delaying the evolution of myocardial necrosis in severely ischemic tissue agents such as verapamil may allow more myocardium to be salvaged by reperfusion. ACKNOWLEDGMENTS The authors gratefully acknowledge assistance of Ms. Nancy Watterson.

the technical assistance of John Tumas and the secretarial

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