Mitochondria Play a Central Role in Nonischemic Cardiomyocyte Necrosis: Common to Acute and Chronic Stressor States

Necrosis

Necrotic cell death is viewed as “dirty” death because the hyperpermeable cell on its terminal trajectory spills intracellular contents (e.g., troponins) which serve as “danger” signals to the innate immune system [54]. The ensuing invasion of inflammatory cells to the site of necrosis, together with recruited myofibroblasts, results in tissue repair and a subsequent reparative fibrosis. This myocardial scarring is a morphologic footprint of previous necrosis while elevated serum troponin levels represent molecular markers of recent cardiomyocyte necrosis.

Foci of fibrosis are found scattered throughout the myocardium of the right and left heart in the explanted failing human heart, where fibrosis is considered the major component to the adverse structural remodeling of myocardium [6]. It follows pari passu that necrosis is a major form of cardiomyocyte loss in the failing heart. The diffuse patchy distribution to myocardial scarring, often most prevalent in the endomyocardium, suggests that cardiomyocyte necrosis is widespread and likely to be an ongoing process. Each episode of necrosis and tissue repair is coupled to myofibroblast-derived collagen fibrillogenesis and the accumulation of stiff type I collagen [89]. This fibrillar collagen, which predominates in scar tissue, has the tensile strength equivalent to steel. In time, the progressive fibrosis of myocardium contributes to diastolic and systolic ventricular dysfunction [90,9,18].

Apoptosis

Apoptosis, on the other hand, is considered a “sterile” form of cell death with cardiomyocytes rapidly engulfed by macrophages never to spill their contents, nor to provoke the immune system or to invoke the invasion of inflammatory cells to the site of injury. Hence, tissue repair eventuating in fibrous tissue does not take place after apoptotic cell death. Accordingly, the apoptotic loss of cardiomyocytes leaves neither a morphologic trail nor a biochemical footprint. Some would question the pathophysiologic role of apoptosis in the loss of cardiomyocytes and the progression to HF [69,81,31,80].

Historical Perspective

Research conducted by Albrecht Fleckenstein and coworkers at the Physiologisches Institute of the University of Freiburg some 40 years ago, led to the novel hypothesis that catecholamine-mediated EICA in cardiomyocytes and mitochondrial Ca²⁺ overloading in particular, combined with ATP depletion, lead to necrosis of these cells. Because the calcitropic hormones, parathyroid hormone (PTH) and vitamin D, potentiate Ca²⁺ overloading and ATP loss, they sensitize cardiomyocytes to catecholamine-induced necrosis [22]. This contrasts to Ca²⁺ channel blockers, cations (e.g., Zn²⁺) which compete with Ca²⁺ entry, or β₁ adrenergic receptor antagonists, each of which were cardioprotective in response to catecholamine treatment of rats using isoproterenol (Isop) [45,36,75,16,76]. The multidisciplinary team of talented investigators from the Institute contributed importantly to this “new principle in cardiac pathophysiology” [21]. They included H. Antonio, H. J. Doering; J. Janke, H. Kammermeier, R. Kaufmann, H. Krause, and H. Tritthart. The Ca²⁺ overloading-to-cardiomyocyte-necrosis hypothesis was extended by these investigators and by Bhattacharya et al. to the hereditary cardiomyopathy of the Syrian Hamster, where the cardioprotective properties of Ca²⁺ channel blockade were likewise demonstrated [51,19,8].

The Fleckenstein hypothesis of nonischemic cardiomyocyte necrosis has recently been broadened into a central mitochondriocentric signal-transducer-effector (MSTE) pathway and found to be common to both acute and chronic stressor states [74,41]. As Fleckenstein envisaged, the signal for this pathway includes catecholamine- or PTH-mediated intracellular Ca²⁺ overloading (see Figure 1). This initial event involves the subsarcolemmal mitochondria spatially closest to Ca²⁺ channels (vis-à-vis the interfibrillar population closer to contractile proteins) [23,41,63,17,11]. The pathway transducer is embodied by induction of oxidative stress within Ca²⁺ overloaded organelles and where their rate of reactive oxygen species generation overwhelms their rate of detoxification by endogenous metalloenzyme-based antioxidant defenses whose activity depends on intracellular Cu²⁺, Zn²⁺ and Se²⁺. A more terminal event and pathway effector is a cyclophilin D-dependent opening of the mitochondrial inner membrane permeability transition pore (mPTP) [34]. The consequent osmotic-based entry of solutes and ongoing swelling accounts for the degeneration of these organelles, loss of ATP synthesis, and ensuing cardiomyocyte necrosis.

How cardiomyocytes determine the activation of apoptotic or necrotic cell death pathways is not fully understood and is under investigation [34,56,60]. The functional roles of these specific pathway components was selectively validated (vide infra) in acute and chronic stressor states using pharmacologic agents that interrupted the cascade at specific downstream targets in rats receiving either Isop or 4 wks aldosterone/salt treatment (ALDOST), respectively [74,73,38,39]. We hypothesize that a lesser level of pathway activation, without ATP depletion, may lead to apoptosis whereas a more severe activation and ATP depletion would result in irreparable damage with cardiomyocyte necrosis and reparative fibrosis in the heart as observed in our animal studies.

Acute Stressor States

Following a single subcutaneous dose of Isop, ionized hypocalcemia is evident within 2 h and attributable to the translocation of Ca²⁺ to diverse tissues that includes the heart, skeletal muscle and peripheral blood mononuclear cells [74]. In cardiomyocytes this EICA includes increased mitochondrial [Ca²⁺]_m coupled with increased H₂O₂ production and increased plasma and cardiac tissue 8-isoprostane, a biomarker of lipid peroxidation, and enhanced propensity to mPTP opening, as evidenced by the response to experimental CaCl₂ stimulation. Intracellular Ca²⁺ overloading and consequent necrosis with myocardial scarring that accompanies Isop-mediated hyperadrenergic state favors the endomyocardium of the left ventricular apex [59,88,64,7,74]. We believe this propensity to catecholamine-induced injury at the apex is in keeping with its greater β₁ adrenergic receptor density [74,7,14] and which is driven by the apical to basal activation of the myocardium needed for the sequential peristaltic-like emptying of the ventricle into the aorta [65,70,10]. These pathophysiologic events were abrogated by pretreatment with either carvedilol, a β-adrenergic receptor antagonist, or quercetin, a flavonoid, each of which have mitochondria-targeted antioxidant properties [74,67,42,72,84]. In transgenic mice with enhanced sarcolemmal L-type Ca²⁺ channel activity, a progressive necrosis of cardiomyocytes is seen together with consequent appearance of reparative fibrosis and pump dysfunction which are accentuated by provocation with Isop [5]. In other rodent models, the loss of cyclophilin D blocked Ca²⁺ overloading-based cardiomyocyte necrosis while overexpression of Bcl-2, an antiapoptotic factor, proved ineffective [57]. Collectively, these findings support the central role of the MSTE pathway in nonischemic cardiomyocyte necrosis during acute hyperadrenergic states.

Chronic Stressor States

Chronic inappropriate (relative to dietary Na⁺) aldosteronism appears with congestive heart failure (CHF), low-renin hypertension, or the adrenal glands’ autonomous production of aldosterone. ALDOST has been used in rats to induce a chronic stressor state to interrogate the specific MSTE pathway components. However, we have been unable to reproduce the expected cardiac remodeling in mice receiving ALDOST. Hence, we have not pursued genetic manipulations to further elucidate the pathway components in mice.

Chronic ALDOST in rats is associated with marked increments in fecal and urinary Ca²⁺ excretion, and which are already evident at wk 1. For the fixed Ca²⁺ intake attendant with laboratory chow provided to these rats, these excretory losses quickly lead to the appearance of plasma ionized hypocalcemia at wk 1 generating a major stimulus to the parathyroid glands’ increased secretion of PTH. The accompanying secondary hyperparathyroidism (SHPT) is further evidenced by bone resorption and a marked fall in bone mineral density and strength seen at wks 4–6 ALDOST [14,15]. PTH-mediated intracellular Ca²⁺ overloading includes both cardiomyocyte cytosolic free [Ca²⁺]_i and subsarcolemmal mitochondrial [Ca²⁺]_m, and is evident at wk 1 and persistent over 4 wks, together with increased production of H₂O₂ by these mitochondria.

This prooxidant state is counterbalanced by an increment in antioxidant defenses which also appears at wk 1. This includes the increased activities of Cu/Zn-superoxide dismutase and glutathione peroxidase and total antioxidant capacity, which consists of nonenzymatic low molecular weight antioxidants [38]. However, the persistent Ca²⁺ overloading of cardiomyocytes present over 4 wks leads to a dysequilibrium between pro- and antioxidants and altered redox state. In the absence of additional increments in endogenous defenses, a marked rise in reactive oxidant species generation by these organelles follows coupled with the subsequent opening of their inner membrane mPTP leading to cardiomyocyte necrosis and consequent tissue repair. Cardiac pathology with microscopic scarring is first evident at 4 wks ALDOST [79,41].

The crucial importance of PTH-mediated Ca²⁺ overloading-induced oxidative stress in leading to the proinflammatory cardiac failure phenotype of this model is evidenced by the upregulation of the redox-sensitive nuclear transcription factor (NF)-κB and the proinflammatory gene cascade it regulates, including ICAM-1, MCP₁ and TNF-α, and which are abrogated by cotreatment with an antioxidant N-acetylcysteine [79]. The importance of NFκB signaling in the heart and its role as a signaling integrator has led some to propose targeting this mediator of cytokine responses in preventing pathologic remodeling with HF [37,26].

The crucial importance of SHPT in mediating this pathophysiologic scenario was demonstrated by a number of interventions. They included: cotreatment with spironolactone, an aldosterone receptor antagonist, which attenuated the marked excretory losses of Ca²⁺ in feces and urine, and thereby prevented ionized hypocalcemia with SHPT [15]; cotreatment with a Ca²⁺ and vitamin D₃-supplemented diet to prevent ionized hypocalcemia and SHPT [25]; surgical parathyroidectomy performed prior to initiating ALDOST [87]; medical parathyroidectomy using cotreatment with a calcimimetic to raise the threshold of the Ca²⁺-sensing receptor of the parathyroids and prevent SHPT [71]; and cotreatment with a Ca²⁺ channel blocker [1]. In subsequent studies the MSTE pathway components were addressed using mitochondria-targeted interventions (see Figure 2). They included quercetin and cyclosporine A, an mPTP opening inhibitor. We found the marked increments in [Ca²⁺]_m and mitochondrial H₂O₂ production, together with increased 8-isoprostane and propensity to their mPTP opening and myocardial scarring seen at wk 4 ALDOST, were each attenuated by cotreatment with either of these agents. Mitochondrial electron transport as a source of reactive oxygen species has also been targeted for cardioprotection [85] while in the vasculature, NADPH oxidase as a source of oxygen radical generation has been targeted [61,49].

Acute Stressor States

Soon after a single subcutaneous dose of Isop, cardiac tissue Ca²⁺ levels rise. Because of cell necrosis, Zn²⁺ transporters are no longer operative and therefore tissue Zn²⁺ levels decline at the site of injury [74,16,76]. In surrounding tissues, however, Zn²⁺ levels rise with the upregulation in its binding protein, metallothionein (MT)-1 [82]. Pretreatment with supplemental ZnSO₄ enhances [Zn²⁺]_i levels and antioxidant potential, and has proven cardioprotective against Isop-induced nonischemic cardiomyocyte necrosis [16,76].

Chronic Stressor States

At 4 wks ALDOST, the expression of Zn²⁺ transporters and MT-1 is upregulated and correspondingly cytosolic and mitochondrial Zn²⁺ levels are increased (see Table 1). In an effort to raise antioxidant defenses, cotreatment with a ZnSO₄ supplement was used. It raised cardiomyocyte [Zn²⁺]_i to 2.0 nM, which was significantly greater than that seen with ALDOST alone (see Table 1) [24,39]. The attendant increased antioxidant capacity of these cells abrogated the rise in mitochondrial H₂O₂ production and levels of 8-isoprostane in cardiac tissue and mitochondria. Moreover, the rise in [Zn²⁺]_i attenuated intracellular Ca²⁺ overloading. Given the attenuation in scarring, expressed as reduced myocardial collagen volume fraction, myocardium was salvaged from necrotic cell death by the ZnSO₄ supplement [38].

The response in [Zn²⁺]_i was examined in rats receiving ALDOST and simultaneously cotreated with either carvedilol or nebivolol [13]. These β₁ receptor antagonists are unique in that they have an additional property as a β₃ receptor agonist, where the latter provokes nitric oxide (NO) formation from endothelial NO synthase which, in turn, releases Zn²⁺ which had been inactive being bound to MT-1 (see Figure 3). In response to carvedilol, [Zn²⁺]_i rose to 2.5 nM and with nebivolol to 2.8 nM, which were greater than that seen with ZnSO₄. The rise in [Zn²⁺]_i activates its sensor, metal-responsive transcription factor (MTF)-1, which upon its translocation to the nucleus upregulates the expression of antioxidant defense genes, including Cu/Zn-SOD, glutathione synthase and MT-1, that collectively raise endogenous antioxidant defenses [38]. Furthermore, the increase in [Zn²⁺]_i gained with either carvedilol or nebivolol served to attenuate Ca²⁺ entry, and consequently [Ca²⁺]_i and [Ca²⁺]_m levels were each reduced. By enhancing endogenous antioxidant capacity and reducing EICA, the multifactorial antioxidant potential of these newer beta antagonists served to markedly attenuate mitochondrial and cardiac tissue 8-isoprostane levels, and the appearance of myocardial scarring [13].

Acute Stressor States

A hyperadrenergic response accompanies acute bodily injury, such as acute myocardial infarction (MI), major surgery including coronary artery bypass surgery, thermal burns, head injury or subarachnoid hemorrhage, and musculoskeletal trauma. Sepsis and diabetic ketoacidosis are systemic inflammatory illnesses accompanied by a hyperadrenergic state.

Nonischemic myocardial necrosis secondary to catecholamine-induced cell death can lead to elevations in serum troponins. These elevations, however, are modest in comparison to the segmental loss of myocardium with an acute reduction in coronary blood flow and MI. Elevated troponins have been reported in acute stressor states that include sepsis, gastrointestinal bleeding, pulmonary embolus and subarachnoid hemorrhage [27,35,53,86]. Nonischemic necrosis is a risk factor for multiorgan failure and mortality associated with ventricular dysfunction [2,91].

Chronic Stressor States

Cardiomyocyte necrosis with elevations in serum troponins indicative of cardiomyocyte necrosis but not due to MI or related to renal failure, are found in patients hospitalized with CHF and are associated with increased in-hospital and overall cardiac mortality [33,50,62,92,52,29,78,32,68,55]. A consistent association between troponin elevation and poor outcomes is found in hospitalized and ambulatory patients with HF. A concentration-dependent relationship exists between the magnitude of troponin elevation and adverse outcomes. Consequently, there is the need for serial troponin measurements to assess changing levels of risk. Increased frequency and magnitude of troponin “leak” results in worsened outcomes and transition from compensated to decompensated HF [43].