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(plateau phase). The increase in extracellular [K^] leads to depolarisation of the

membrane potential and a reduction in the duration and amplitude of the action

potential (Carmeliet, 1978) and is thought to result in decrease of developed tension in

ischaemia. However, only a slight decrease in developed tension associated with

elevated (up to 12mM) extracellular [K^] was observed in isolated cat ventricular

muscle preparations (Kavaler et al, 1972), indicating that the membrane potential

changes associated with increased extracellular [K^] contribute little to the rapid fall of

tension in ischaemia. Inspite of the increase in extracellular [K^] during ischaemia, there

is no indication of a reduced activity or inhibition of the Na^ /K^-ATPase. In isolated

globally ischaemic guinea pig hearts, Kléber (1983) demonstrated that intracellular [Na“^]

was unchanged after 15 min. of ischaemia. Subsequently, Wilde and Kléber (1986)

confirmed this using isolated guinea pig papillary muscle preparation subjected to

conditions which mimicked ischaemia (such as hypoxia, increased extracellular [K^],

extracellular acidosis and absence of substrate).

The rapid decrease in developed tension that occurs during anoxia, when glycolysis is

accelerated, does not seem to be attributable to changes in action potential duration.

rapid fall in tension over 5 min. anoxia in the presence of 5mM glucose but the reduction

in action potential duration was less than 5% of control which decreased to 40% with

prolonged period of anoxia (60 min.).

In contrast, some studies suggest that with inhibition of glycolysis, either by glycogen

depletion in ferret ventricular muscle or by 2-deoxyglucose treatment in guinea pig

ventricle (Allen and Orchard, 1987), anoxia leads to shortening of the action potential

duration to less than 20% of control within 2-3 min. along with a rapid fall of tension to

0-5% of control. Such rapid and dramatic changes would be expected to lead to reduced

Ca^^ release from the SR or failure of Ca^^ delivery to the contractile proteins of the

myofibrills and may explain the reduced Ca^"*" transients reported during early anoxia

when glycolysis was prevented (Allen and Orchard, 1983). Based on this study, failure

of Ca^^ delivery to the contractile proteins (e.g., troponin, actomyosin-ATPase) during

ischaemia has been implicated to contribute to the rapid decline of tension, but the

mechanism of this failure in Ca^^ delivery has not been fully resolved.

A decrease in intracellular pH (acidosis due to accumulation) during

ischaemia/hypoxia may also contribute to the observed decrease in tension. Increase in

glycolysis and lactate production in myocardial anoxia (Williamson, 1966) led to the

suggestion (Katz and Hecht, 1969) that the resultant intracellular acidosis was

responsible for the decrease of tension during ischaemia. However, more recent

n.m.r. measurements of intracellular pH in isolated hearts during ischaemia and hypoxia

have shown that the contribution of acidosis is too small and too slow to explain the

rapid decline in tension (Jacobus et al, 1982; Allen et al, 1985). Jacobus et al (1982)

showed that the decrease in mechanical function during ischaemia was greater than

acidosis after several minutes, whereas developed tension decreased rapidly (2-3 min.).

Moreover, with inhibition of glycolysis, hypoxia led to a rapid fall in tension without any

acidosis (Allen et al, 1985).

Accumulation of inorganic phosphate (Pi) as a result of high energy phosphate

degradation during ischaemia and hypoxia is probably the most important contributor of

early contractile failure of the myocardium. Experiments on the e ^ c t of Pi on skinned

cardiac fibres have shown that Pi has a pronounced inhibitory effect on maximum Ca^'*’-

activated tension and in addition, leads to a reduction in Ca^^ -sensitivity of the

myofibrils (Allen and Orchard, 1987). During the first few minutes of

hypoxia/ischaemia, while [ATP] remains relatively constant, [PCr] falls considerably

resulting in a substantial increase in [Pi] (see Section 1.B.2). Increased intracellular [Pi]

can translate a relatively small decline in high energy phosphate into a major effect on

contractile activity. For example, a small fall in PCr (20% of control) during ischaemia

can cause an increase in [Pi] from l-2mM normoxic to about 20mM which in turn results

in reduction of the maximum Ca^^-activated tension development to 50% of control. In

addition, through its effect on sensitivity of the myofibrils to Ca^"^, increased [Pi] further

reduces developed tension to 2 0% of control.

In conclusion, several parameters may be responsible for the early contractile failure

observed during ischaemia/hypoxia and additional experimental evidence is required

for the understanding of the mechanism underlying myocardial contractile failure.

l.C M e ta b o lis m D u rin g R ep erfu sio n

A number of studies have shown that myocardial function remains depressed and

recovers slowly following reperfusion of the reversibly damaged myocardium (Weiner

1982). The precise mechanisms responsible for this dysfunction are not fully understood,

but could be potentially related to prolonged depletion of ATP.

Reperfusion or reoxygenation of the myocardium in vitro/in vivo restores PCr content to

or above pre-ischaemic/pre-anoxic levels very rapidly but repletion of ATP is much

slower (Reibel and Rovetto, 1978; De Boer et al, 1980; Reimer et al, 1981; Swain et al, 1982;

Takeo and Sakanashi, 1983). Similar observations have also been reported using ^^P

n.m.r. spectroscopy on Langendorff perfused rat hearts (Bailey and Seymour, 1981; Sako

et al, 1988).

Reimer et al (1981) demonstrated that after 24 hrs. reperfusion of ischaemic dog heart,

ATP was significantly depressed and at only half of the normal content and remained

significantly reduced even after 4 days of reperfusion. This prolonged depression in the

ATP concentration was not the result of a lowered ability to rephosphorylate AMP or

ADP, but rather can be attributed to the significantly reduced total adenine nucleotide

pool after 24 hrs. as well as after 4 days of reperfusion. Such persistent depression in the

total adenine nucleotide pool can cause an extra risk since a critically low ATP

concentration will be reached earlier during the next ischaemic attack (Kloner et al, 1981;

Geft et al, 1982). Since the major ATP-utilising reaction in the myocardium is the actin-

myosin ATPase, the depressed myocardial contractility following reperfusion may reflect

this reduced availability of ATP.

There are a number of potential explanations for delayed repletion of ATP.

Mitochondrial respiration being the major route for ADP phosphorylation under aerobic

conditions, damage to the mitochondrion or limitation in substrates for oxidative

phosphorylation during reperfusion could account for a reduction in the rate of ATP

supranormal levels (see above) argues against a defect in mitochondrial oxidative

phosphorylation as the limiting reaction in ATP repletion. N o evidence of ADP or AMP

accumulation during reperfusion has been found (Reimer et al, 1981; Swain et al, 1982).

Moreover, adenylate charge was rapidly restored on reperfusion following brief

coronary occlusion in the open chest dog (Swain et al, 1982). In contrast. Hardy et al

(1991) have shown a specific lesion of the NADHiCoQ reductase (Complex I) in

mitochondria isolated immediately after reoxygenation of hypoxic (40 min.) perfused rat

heart. Such a defect in the respiratory chain would lead to a lowered ability to synthesise

ATP and therefore delay functional recovery of the myocardium.

Mallet et al (1990) have shown that a high cytosolic phosphorylation potential and

functional glycolysis are required to prevent de-energisation and contractile failure

during reperfusion of the working guinea pig heart subjected to 25 min. global low-flow

ischaemia and norepinephrine infusion. Reperfusion (30 s.) with 5mM glucose +

pyruvate caused a nearly 3-fold increase in the 3-phosphoglycerate/glyceraldehyde-3-

phosphate ratio along with a rise in the dihydroxy acetone phosphate/glycerol-3-

phosphate ratio, together with an increase in the ATP content and decreases in Pi

concentration and adenosine plus inosine release. These changes indicate a shift in the

glyceraldehyde-3-phosphate/phosphoglyceratekinase reaction towards higher cytosolic

NAD'^/NADH ratio and ATP potential. Reperfusion with either glucose or pyruvate as

sole substrates, failed to restore phosphorylation potential and contractile function to

pre-ischaemic level.

The delayed repletion of the adenine nucleotide pool on reperfusion may in part be due

to the limited availability of precursors for the de novo or salvage pathways for adenine

nucleotide synthesis, since these would be washed out from the myocardium on

Figure 1.2

Purine salvage and degradation pathways in myocytes and endothelial cells.

M y o cy te