Inflammation and oxidative stress produce ROS/RNS through a number of enzymatic systems including XO. It is well known that mitochondria are both targets and sources of oxidative stress, which we have shown in VO results in inhibition of the respiratory chain. This is important since VO produces an increase in pressure-volume area (PVA) as shown in , which is known to require higher myocardial oxygen consumption (MVO2
) and ATP consumption (15
). We reasoned that this combination of increased energy demand and mitochondrial dysfunction increases the susceptibility of the VO heart to failure. Indeed, we have recently established a novel interaction between bioenergetics and activation of MMPs in the cardiomyocyte of the VO heart (16
). This is particularly interesting in the context of XO since its substrates, xanthine and hypoxanthine, are elevated under increased bioenergetic demand and bioenergetic dysfunction. In the current study, we report that VO causes an increase in XO activity in LV tissue and cardiomyocytes without changes in total XO protein, consistent with an oxidative post-translational activation of XO ().
Since the activation of XO through this mechanism involves the oxidation of thiols, we reasoned that mitochondrial derived oxidants are an early event that could lead to the activation of XO. To test this hypothesis, MitoQ is used to inhibit mitochondrially-derived ROS in the stretched cardiomyocytes. Indeed, MitoQ prevents both stretch induced-XO activation and mitochondrial swelling and disorganization (). These results suggest that mitochondria, which comprise 40% of the cardiomyocyte by volume, may be an important source of ROS and play a regulatory role in XO activation in the VO cardiomyocyte.
The current study also demonstrates loss of myofibrillar integrity in isolated cardiomyocytes subjected to cyclic stretch. We have previously shown that 24 hours of ACF results in increased TNF-α levels (35
) and ROS formation and matrix metalloproteinase (MMP) activation (16
) within cardiomyocytes. Cytokines, XO, and ROS have been shown to cause MMP activation (36
) and there is increasing evidence that cardiomyocyte MMP activation is responsible for myosin and troponin degradation during cardiac ischemia reperfusion injury (39
). In addition, transgenic mice expressing active MMP-2 driven by the α-myosin heavy chain promoter exhibit breakdown of Z-band registration, lysis of myofilaments, and disruption of sarcomere and mitochondrial architecture (41
). It is of interest that we have recently demonstrated extensive cardiomyocyte myofibrillar loss in association with increased oxidative stress in the myocardium of VO patients with chronic isolated mitral regurgitation (3
). Thus, it is tempting to speculate that mitochondrially derived ROS and XO-mediated MMP activation may play a causative role in the myofibrillar degeneration that has now been identified in the rat (16
), dog (42
), and human (3
) with isolated VO.
Increased cardiomyocyte XO activity with acute ACF is also associated with decreased State 3 maximal bioenergetic capacity of isolated subsarcolemmal mitochondria, which is normalized by allopurinol (). Mechanical stretch is associated with increased cardiomyocyte XO activity and abnormal mitochondrial structure that is prevented by allopurinol and MitoQ. NADPH oxidase and uncoupled NOS activation have also been identified in cardiomyocyte stretch (44
). The current study demonstrates XO activation in a heart failure model may be a direct response to physical stretch. Further, our in vitro
studies also implicate the mitochondria as a source of ROS by demonstrating that XO activation and mitochondrial and cytoskeletal derangements with stretch can be prevented by MitoQ. Therefore, it is tempting to speculate that XO activation is related to ROS production from mitochondrial structural alterations and that allopurinol and MitoQ may have synergistic effects in vivo
. These data do not exclude a role for NADPH oxidase and iNOS in contributing to the VO-dependent response to stretch but they do suggest that they are required to interact with both mitochondria and XO to contribute to the pathology.
The in vivo
and in vitro
findings of the current study suggest that increased XO activity and mitochondrial oxidative stress are central factors in bioenergetic dysfunction in the face of the increased ATP requirements and MVO2
of VO. Studies by Hare and coworkers have shown that acute administration of allopurinol decreases the MVO2
while improving contractile function in patients with dilated cardiomyopathy (46
) and in dogs with pacing tachycardia induced heart failure (47
), suggesting improved myocardial efficiency. We further speculate that increased levels of ADP and AMP, particularly in the setting of mitochondrial dysfunction, that are degraded to XO substrates hypoxanthine and xanthine, can set up a self-perpetuating cycle by which activated XO produces ROS that damage mitochondria, that in turn causes further ROS production and XO activation (). In support of this argument, it is of interest that the acute stretch of VO causes relatively greater XO activation in isolated cardiomyocytes (300%) than in the LV tissue homogenate (15%).
Increased xanthine oxidase activity leads to mitochondrial dysfunction in the VO heart
LV ejection fraction is preserved after 24 hours of ACF. However, the LV ESPVR, which provides a load independent index of LV contractility, is depressed in the acute 24 hour ACF, and allopurinol improves both LV contractility and diastolic function (). It is of interest that increased cardiac ADP levels have been linked to diastolic dysfunction by outcompeting ATP at the actin-myosin crossbridge site and subsequently impairing the relaxation process by delayed ADP dissociation, which is the rate limiting step in cross bridge cycling (48
). Indeed, artificially altered ADP levels have been shown to directly correlate with increased LVEDP in the rat heart (49
). The beneficial effect on LVED σ is particularly important because wall stress is the driving force for LV hypertrophy in VO (1
). Because LV mitochondrial and diastolic function are simultaneously normalized by allopurinol, it is tempting to speculate that allopurinol attenuates ROS-dependent mitochondrial damage and improves diastolic function by improving maximal ADP-stimulated respiratory capacity and decreasing buildup of ADP.
Whether allopurinol improves LV function in acute VO through its effect on mitochondrial respiration cannot be conclusively demonstrated in the current study, since we only measured the respiration of sub-sarcolemmal mitochondria respiration and not intermyofibrillar mitochondrial respiration. In addition, in vitro
studies demonstrate that XO depresses myofilament sensitivity to calcium and that it co-localizes with nitric oxide synthase-1 in the sarcoplasmic reticulum in the mouse cardiomyocyte, which can regulate excitation-contraction coupling as well as myofilament oxidative damage (50
). Specifically, XO inhibition restores ryanodine receptor nitrosylation, reverses diastolic sarcoplasmic reticulum calcium leak, and improves cardiomyocyte contractility in the spontaneously hypertensive heart failure rat (52
) and improves the maladaptive changes in calcium cycling proteins associated with LV failure in the pacing tachycardia dog model (53
). In the current study, XO protein staining by immunohistochemistry is most concentrated at the Z-line in the cardiomyocyte, which is similar to the findings in the human cardiomyocyte (3
). Further, in vitro
stretch of cardiomyocytes results in loss of myofibrillar structural integrity of the Z-line concurrent with increase XO activity, both of which are prevented with allopurinol or Mito Q. Thus, we cannot rule out an alternative calcium/myofilament mediated mechanism by which allopurinol improves LV contractile performance in this acute VO.
In summary, we have shown that acute VO increases XO activity in heart tissue and isolated cardiomyocytes and that defects in subsarcolemmal mitochondrial respiration and LV dysfunction with VO are reversed by allopurinol. Further, cyclic stretch of cardiomyocytes increases XO activity producing mitochondrial structural defects that are attenuated by allopurinol or MitoQ. Taken together, these studies indicate that XO activation from stretch induced oxidative stress may be central to both bioenergetic and LV dysfunction in acute VO.