Phosphate toxicity and SERCA2a dysfunction in sudden cardiac arrest

Almost half of the people who die from sudden cardiac arrest have no detectable heart disease. Among children and young adults, the cause of approximately one‐third of deaths from sudden cardiac arrest remains unexplained after thorough examination. Sudden cardiac arrest and related sudden cardiac death are attributed to dysfunctional cardiac ion‐channels. The present perspective paper proposes a pathophysiological mechanism by which phosphate toxicity from cellular accumulation of dysregulated inorganic phosphate interferes with normal calcium handling in the heart, leading to sudden cardiac arrest. During cardiac muscle relaxation following contraction, SERCA2a pumps actively transport calcium ions into the sarcoplasmic reticulum, powered by ATP hydrolysis that produces ADP and inorganic phosphate end products. Reviewed evidence supports the proposal that end‐product inhibition of SERCA2a occurs as increasing levels of inorganic phosphate drive up phosphate toxicity and bring cardiac function to a sudden and unexpected halt. The paper concludes that end‐product inhibition from ATP hydrolysis is the mediating factor in the association of sudden cardiac arrest with phosphate toxicity. However, current technology lacks the ability to directly measure this pathophysiological mechanism in active myocardium, and further research is needed to confirm phosphate toxicity as a risk factor in individuals with sudden cardiac arrest. Moreover, phosphate toxicity may be reduced through modification of dietary phosphate intake, with potential for employing low‐phosphate dietary interventions to reduce the risk of sudden cardiac arrest.


| INTRODUCTION
Sudden cardiac arrest occurs without warning when an individual's heart suddenly stops beating. 1 In sudden cardiac death, closely related to sudden cardiac arrest, "nine out of 10 people who have a cardiac arrest outside of a hospital dieoften within minutes". 2 Yet, nearly half of individuals with sudden cardiac arrest have no detected "coronary or structural heart disease". 3 Among children and young adults, the cause of approximately one-third of sudden cardiac deaths remains unexplained after thorough examination. 4,5 Consequently, "unexplained sudden cardiac death is often attributed to cardiac arrhythmia caused by cardiac ion-channel dysfunction, which is undetectable in a conventional autopsy". 5 In a normal heart, contractility is controlled through various channels and pumps that handle changes in intracellular calcium levels-balancing the influx and efflux of calcium ions across membranes with each heartbeat. 6 However, cellular accumulations of dysregulated inorganic phosphate, a condition known as phosphate toxicity, can disrupt homeostasis of major organ systems, including the cardiovascular system. 7 The present perspective paper proposes a pathophysiological mechanism by which phosphate toxicity disrupts and halts normal calcium handling in the heart, leading to sudden cardiac arrest (SCA).

| MATERIALS AND METHODS
The aim of the present perspective paper is to propose novel insights and future directions for research in the pathophysiology and cause of SCA associated with phosphate toxicity. A Grounded Theory Literature-Review method was used to add rigor and objectivity when researching the paper. 8,9 Beginning with a literature review, keywords relevant to research on SCA, cardiac physiology, and phosphate toxicity were searched in Google, Google Scholar, Pub Med, Scopus, and other databases. Research findings selected from the literature were used as data for analysis in the grounded theory study, and data were categorized into concepts for comparative analysis. As more data were acquired, specific concepts were grouped into larger themes, and relationships of themes were eventually synthesized into a final explanatory theory grounded in evidence from the research literature. The grounded theory was then used to present a proposed association of SCA with phosphate toxicity.

INORGANIC PHOSPHATE
Adult patients who were resuscitated from cardiac arrest were found to have high serum phosphate levels following restoration of spontaneous circulation (ROSC), and worse outcomes were associated with elevated serum phosphate in the patients at hospital discharge. 10 An association of high serum phosphate with out-of-hospital cardiac arrest was also found in 105 patients in an earlier case-control study, 11 and higher levels of serum phosphate in people with prior myocardial infarction were independently associated with increased risk of heart failure, cardiovascular events, and all-cause death. 12 A more recent German study of cardiac arrest patients with ROSC found that higher risk of mortality was predicted by initial serum phosphate levels greater than 2.7 mmol/L. 13 Additionally, dysregulated phosphate homeostasis is common in patients with chronic kidney disease (CKD), 14 and sudden cardiac death accounts for 40% of all deaths in CKD, rising to 78% in end-stage renal disease. 15 Metabolic derangement in cancer patients increases risk of cardiac arrest with low survival rate, 16 which may be mediated by phosphate toxicity associated with overgrowth of cancer cells. 17 Additionally, tumor lysis syndrome during cancer treatment releases large amounts of Pi from tumors, which is associated with arrythmia and in-hospital mortality, 18 and sudden cardiac death increases during cancer treatment. 19 Cases of cardiac arrest following acute hyperphosphatemia have also been reported in accidental infusion with potassium phosphate 20 and administration of sodium phosphate. 21 "Hyperphosphatemia or even serum phosphate levels within the 'normal laboratory range' are highly associated with increased cardiovascular disease risk and mortality in the general population," including distinct mechanisms for vascular calcification, hypertension, myocardial fibrosis, cardiac valvular calcification, atherosclerosis, and left ventricular hypertrophy. 22 Yet, no studies have investigated the pathogenesis of SCA associated with phosphate toxicity-a condition caused by abnormal tissue accumulation of dysregulated inorganic phosphate (Pi). 7 Furthermore, because SCA occurs in many cases without evidence of cardiovascular disease, an association of SCA with phosphate toxicity likely involves a novel pathophysiological mechanism that operates independently of other cardiovascular conditions induced by hyperphosphatemia.
Dysregulated phosphorus metabolism leading to phosphate toxicity may result from excessive dietary phosphate intake from food, or from Pi additives in food and beverages, in combination with insufficient renal excretion. 23 Serum Pi is normally regulated by a sensitive network of hormones from an axis formed by the kidneys, intestines, parathyroid glands, and skeletal system. 24 Excessive serum levels of Pi are lowered as parathyroid hormone from the parathyroid glands and FGF23 from bone (with cofactor Klotho) reduce Pi reabsorption in the kidneys and increase phosphaturia. Pi intestinal absorption is also increased by kidney biosynthesis of the bioactive form of vitamin D, 1,25(OH) 2 D 3 (calcitriol). Conversely, reduced calcitriol biosynthesis in the kidneys lowers intestinal Pi absorption and reduces serum Pi levels. Moreover, serum Pi levels can appear normal, even though intracellular Pi concentrations are high with effects of phosphate toxicity. 25 Hypophosphatemia can also occur in emergency conditions as large amounts of extracellular Pi are rapidly shifted into cells. 26 The present paper presents evidence inferring that phosphate toxicity inhibits function of the sarcoendoplasmic reticulum calcium ATPase 2a (SERCA2a) pump in cardiac muscle. The SERCA2a isoform, a P-type of pump in cardiac cells, has almost identical enzyme reactions with the SERCA1a isoform in fast-twitch skeletal muscle. 27 Similarly, SERCA2a regulates intracellular calcium (Ca 2+ ) in the sarcoplasmic reticulum 28 -a specialized form of endoplasmic reticulum. 29 SERCA2a itself is regulated within a narrow physiological range by phospholamban and other micropeptides, which help reduce cardiac contractility during resting conditions. 27

| SARCOPLASMIC RETICULUM AND CA 2+
A transient increase in intracellular calcium concentrations within skeletal and cardiac muscle fiber is controlled by calcium release and reuptake within the sarcoplasmic reticulum (SR). In excitation-contraction coupling, 30-32 an action potential arriving at the muscle cell membrane, the sarcolemma, produces a small influx of calcium through L-type Ca 2+ channels in the T-tubule. Through a process called calcium-induced calcium release, a large efflux of Ca 2+ from the SR is subsequently released into the cytoplasm through the ryanodine receptor 1 (RyR1) in skeletal muscle and ryanodine receptor 2 (RyR2) in cardiac muscle. The efflux of Ca 2+ throughout the muscle fiber binds to troponin C and alters the position of tropomyosin, allowing engagement of a cross-bridge mechanism between myosin and actin filaments. Adenosine triphosphate (ATP) is used to power the cross-bridge mechanism causing muscle contraction. Muscle fiber contraction ends and fiber relaxation begins with Ca 2+ reuptake from troponin into the SR through active transport of Ca 2+ by SERCA pumps powered by ATPase, 33 allowing tropomyosin to once again block the engagement of myosin and actin filaments. During ATP hydrolysis, adenosine diphosphate (ADP) and Pi are released as end products. Figure 1 is a schematic drawing of the SR in cardiac muscle.
A key finding from earlier research on skeletal muscle is that fiber relaxation is delayed if Pi accumulates in the fiber, which slows Ca 2+ reuptake by the SR. 35 This delay of fiber relaxation by accumulated Pi appears to be an example of inhibition, which is defined chemically as "the stopping or slowing of the rate of a chemical reaction". 36 Although enzyme systems normally rely on end-product inhibition to regulate homeostasis, the inhibition of ATP hydrolysis that powers the Ca 2+ pumps within the SR of skeletal muscle is likely a pathophysiological disturbance associated with phosphate toxicity from rising concentrations of Pi in the tissues of the body. A milder type of delayed muscle relaxation commonly occurs in overexertion of skeletal muscle contraction, which disrupts Ca 2+ release and reuptake by the SR 37 and produces temporary muscle cramping or tetany as Pi accumulates in the muscle fiber when large amounts of ATP are hydrolyzed to power the cross-bridge mechanism. 38 Importantly, a major challenge in measuring an inhibitory effect of Pi during myocardial fiber relaxation in the past has been that the Pi signal is often obscured by ventricular blood pooled in the active heart. 39

INHIBITION OF CA 2+ UPTAKE
In both skeletal and cardiac muscle, the ATPase enzyme of the SERCA pump cycles through two conformational states during Ca 2+ uptake. 40 In the E 1 state, 2 Ca 2+ cations in the muscle cytoplasm attach to the ATPase enzyme in combination with an ATP molecule, forming an intermediate (E 1 Ca 2 ATP). ATP hydrolysis releases ADP into the cytoplasm and actively transports the intermediate across the SR membrane in the E 2 state (E 2 Ca 2 P). As the luminal portion of the transmembrane enzyme opens, the two occluded Ca 2+ cations are released into the SR lumen in exchange for two (or three) protons (H + ) acquired by the intermediate (E 2 H 2 P). The intermediate is then dephosphorylated (E 2 H 2 ), releasing Pi into the cytoplasm (note that the phosphorylation P domain of ATPase lies on the cytoplasmic portion of the enzyme), followed F I G U R E 1 Sarcoplasmic reticulum in cardiac muscle. Adapted from Periasamy et al. 34 by dehydrogenation which releases the occluded protons into the cytoplasm as the pump returns to the E 1 state.
To prevent cellular ATP depletion, organisms inhibit ATP hydrolysis with specific mechanisms, which is desirable to protect against wasteful ATP hydrolysis that precipitates cellular death. 41 In addition, a more general mechanism occurs as "ATP hydrolysis is inhibited by its products, ADP and/or phosphate". 42 End-product inhibition of ATP hydrolysis in the SERCA2a pump implies that high amounts of dysregulated Pi may reduce critical energy sources from ATP in pathological conditions like sudden cardiac arrest. Unfortunately, "energy-related dysfunctions have not been widely seen as causes of common diseases, although evidence points to such a link for certain disorders," such as mitochondrial dysfunction that affects many organ systems. 43 Yet, lacking ATP hydrolysis for energy production in SERCA2a pumps, calcium ions released from the sarcoplasmic reticulum (SR) during initiation of cardiac muscle contraction cannot be actively transported back into the SR, potentially leading to cardiac arrhythmias and other abnormalities including SCA and death.
Evidence supporting the inhibition of ATPase hydrolysis by Pi in cardiac dysfunction has gradually accumulated over the years. In related research from 1974 on the sodium-potassium-ATPase pump (Na-K-ATPase), Garay and Garrahan found that increased intracellular ADP and Pi concentrations inhibited the pump in red cells and that "the most likely mechanism for the inhibitory effect of phosphate is the reduction of the net rate of dephosphorylation of the Na pump". 44 Schmidt-Ott et al in 1990 first reported that Pi at 10 mM inhibited ATPase activity in human myocardium fibers. 45 Xiang and Kentish noted in 1995 that "an increase in intracellular Pi reduces sarcoplasm reticular calcium loading". 46 Hajjar et al examined human myocardium and found that increased Pi concentrations decreased the development of maximum contractile force by 56% in non-failing myocardium and by 36% in failing myocardium with reduced myofibrillar ATPase activity. 47 In 2004, Tomaselli and Zipes wrote that SERCA2a was a primary remover of calcium (Ca 2+ ) from cytoplasm and that "aberrant Ca 2+ handling is a recurrent theme in sudden death from heart failure". 48 More than 30 years after related research on ADP and Pi in the Na pump, Periasamy and Kalyanasundaram noted in 2006 that factors including "ADP and inorganic phosphate level can influence SERCA pump activity". 49 By 2008, Wu et al hypothesized that Pi is "the most significant product of ATP hydrolysis in limiting the capacity of the heart to hydrolyze ATP in vivo". 50 The researchers estimated baseline cytosolic Pi concentration in the canine myocyte at approximately 0.29 mM, which increased to 2.3 mM at near maximum cardiac oxygen consumption. Tewari et al noted that low Pi and ADP concentrations are necessary to avoid altering the energetic metabolic state of the myocardium and impairing normal cardiac mechanical function. 51 Lopez et al stated that "physiological rates of myocardial ATP consumption require the heart to resynthesize its entire ATP pool several times per minute," and the researchers hypothesized that altered concentrations of phosphate metabolites (Pi, ATP, and ADP) "have direct roles in impeding contractile function of the myocardium". 52 Using a rat model with transverse aortic constriction, the researchers calculated that increased Pi impaired the ATPase myosin crossbridge mechanism compared to controls.
Recently, Valkovič et al measured myocardial Pi in vivo using 7T 31 P-cardiovascular magnetic resonance spectroscopy, finding higher Pi/ATP ratios in patients with cardiomyopathy compared to healthy controls. 39 Wakefield et al noted that cytosolic Pi concentrations in the rat myocardium are expected within the range of 2-8 mM. 53 The researchers suggested that increasing Pi concentrations up to 5 mM assist in the detachment of myosin during myofilament lengthening-induced relaxation in adult female Wistar rats. However, Pi-assisted myofilament relaxation is unrelated to end-product inhibition of ATPase from higher concentrations of dysregulated Pi, which burdens reuptake of calcium ions in the SR. For example, Kentish studied myoplasmic Pi concentrations in skinned rat heart muscle and found that a range up to 30 mM Pi greatly inhibited maximum isometric force development. 54 Wakefield et al further noted that "monitoring cytosolic Pi while also measuring myocardial function is not yet technically feasible", 53 and newer technologies to measure myocardial Pi in vivo may lead to further discoveries in the association of phosphate toxicity with SCA. Relatedly, the sodium calcium exchanger (NCX) in the myocardium is also an ATP-dependent ion channel, and Wakefield et al inferred that elevation of cytosolic Pi by the Na + / Pi cotransporter Pit2 would also elevate cytosolic Na + , subsequently inhibiting NCX activity and elevating intracellular calcium. 53 In addition to SCA, inhibition of SERCA2a function by Pi may contribute to pathogenesis of heart failure: "During heart failure, one of the most pronounced cellular changes is an increase in end-diastolic cytosolic calcium levels and prolongation of the calcium transient during diastole. This is primarily due to a decrease in SR calcium uptake because of SERCA2a dysfunction". 55 "Heart failure patients are predisposed to develop arrhythmias", 56 and future research should investigate phosphate toxicity and SERCA2a dysfunction in arrhythmogenesis. Xie et al attributed arrhythmia in cardiomyopathy to the transfer of mitochondrial calcium to the SR, and the researchers reduced arrhythmia in a murine model by knocking down SERCA2a gene expression. 57 However, follow-up investigations of this finding should explore the confounding role that phosphate toxicity might play by inducing cardiomyopathy. 58 For example, upregulated expression of SERCA2a in cardiomyopathy improves contractility and cardioprotection, 59 but increased SERCA2a expression without restored phosphate homeostasis may also increase the effects of SERCA2a dysfunction leading to arrhythmia. By comparison, downregulating dysfunctional SERCA2a in cardiomyopathy would reduce arrhythmia, but also lower contractility and cardioprotection.
Cytosolic Pi is regulated in the myocardium by the Pit2 Na + /Pi cotransporter, 53 and Pit2 is upregulated by the epsilon isoform of protein kinase-C (PKC-ε). 60 Importantly, long-term expression of the delta isoform of protein kinase-C (PKC-δ) "can damage cardiomyocytes, and PKC-δ can significantly downregulate sarcoplasmic reticulum Ca 2+ ATPase (SERCA2) gene expression, and reduce myocardial contractility", 61 an effect potentially related to increased cytosolic Pi in the myocardium. Of relevance, SERCA2a gene expression is downregulated in chronic heart failure (CHF), and, as previously mentioned, CHF is associated with elevated serum phosphate levels. 12 Patients with hypertrophic cardiomyopathy (HCM) have increased risk of sudden cardiac death and arrhythmias, and further investigations of novel risk factors are needed. 62 A recent case study reported a patient with HCM presenting with secondary hypoparathyroidism, which the authors associated with the effects of hyperphosphatemia and elevated levels of calcium phosphate product and FGF23. 63 Further clinical and epidemiological investigations of dysregulated serum Pi as a novel risk factor associated with HCM are warranted, including population studies of dietary phosphate exposure, animal models, and in vitro and in vivo experiments that illuminate the effects of myocardial Pi in arrhythmia and sudden cardiac arrest.
Seidlmayer et al noted that polyphosphate (polyP), a chain of inorganic orthophosphates linked by phosphoanhydride bonds, is in high demand in metabolically active tissue in the brain and heart, and polyP generation and metabolism is associated with stress conditions such as ischemia and reperfusion. 64 The researchers found that short-chain polyP activated the mitochondrial permeability transition pore (mPTP), which ultimately causes cardiomyocyte death. Short-chain polyP was also associated with mitochondrial uncoupling/dysfunction and metabolic failure in ATP production. These polyP effects in the myocardium could impact SERCA2a function. Other effects of mitochondrial dysfunction include calcium accumulation in the presence of phosphate 65 and increased reactive oxygen species related to hyperphosphatemia. 66 Additionally, Abbasian and Harper found that high extracellular concentrations of Pi increased Pi transport and formed polyP in blood platelets. 67 A similar mechanism may generate polyP in cardiomyocytes from dysregulated levels of Pi and should be investigated. Figure 2 summarizes the causative pathway in which the association of phosphate toxicity with SCA, the dotted arrow, is mediated by end-product inhibition of SERCA2a ATP hydrolysis by Pi. Further research is needed to confirm this pathophysiological mechanism as a risk factor for sudden cardiac arrest. Importantly, research may demonstrate that rising levels of myocardial Pi during or following exercise can increase risk of SCA associated with phosphate toxicity in young people 68 and also in older patients with underlying cardiovascular disease.

RESEARCH
Other areas for future research are briefly mentioned here. Kidney disease is associated with epilepsy 69 and end-stage renal disease is associated with type 1 diabetes. 70 Inferences from these associations suggest that dysregulated Pi from impaired renal function might cause SERCA2a dysfunction related to sudden unexpected death in epilepsy (SUDEP), 71 and Pi-induced SERCA2a dysfunction may be associated with dead-in-bed syndrome in type 1 diabetes. 72 More research of the role of phosphate toxicity in these associations is needed.
Glycogen phosphorylase isoenzyme BB (GPBB) in myocardial tissue increases Pi attachment to glucose F I G U R E 2 The association of phosphate toxicity with sudden cardiac arrest is mediated by end-product inhibition of SERCA2a ATP hydrolysis by Pi.
(glucose-1-phosphate) during glycogenolysis, and GPBB is released into circulation during the first few hours following myocardial infarction. 73 Future research should explore the present author's proposed hypothesis that emergency intracardiac injections of GPBB may lower Pi levels in SCA, possibly alleviating end-product inhibition of SERCA2a ATP hydrolysis.
A murine model of CKD found that sevelamer carbonate, which reduces phosphate intestinal absorption, improved cardiovascular abnormalities including aortic stiffness, diastolic dysfunction, and left ventricular hypertrophy. 74 However, in a randomized controlled trial of patients with CKD, compliance with prescribed sevelamer carbonate was poor and no cardiovascular effects were found compared to placebo. 75 Evidence is lacking to suggest that sevelamer carbonate reduces arrhythmias or SCA in humans with CKD or in murine models. Phosphate toxicity may also be reduced through modification of dietary phosphate intake, 76 with potential for employment of low-phosphate dietary interventions to reduce the risk of SCA. Additionally, cardiovascular disease, renal dysfunction, and premature death are associated with consumption of Pi additives in ultraprocessed foods, 77 which is another factor potentially contributing to SCA that requires further investigations.
Diammonium phosphate is added to tobacco products to improve flavor, 78 and tobacco plants are often grown with triple superphosphate fertilizer. 79 Coincidently, among patients with CKD, smokers had higher serum phosphate levels compared to non-smokers, even after researchers adjusted for renal function, 80 inferring that tobacco smoking might increase phosphate exposure through inhalation. Hypothetically, as a potential risk factor for SERCA2a dysfunction, excessive inhalation of Pi from tobacco smoke could explain why a meta-analysis found a threefold increased risk of sudden cardiac death in current smokers compared to never smokers. 81 Further investigations are warranted linking phosphate in tobacco with cardiovascular disease and cancer.

| CONCLUSION
Energy-related dysfunctions are not widely considered as causes of common diseases. Evidence reviewed in this paper supports the proposal that end-product inhibition of SERCA2a in the heart SR occurs as increasing levels of Pi from ATP hydrolysis bring cardiac function to a sudden and unexpected halt in SCA. The present paper concludes that end-product inhibition of ATP hydrolysis by rising levels of Pi is a mediating factor in the association of SCA in individuals with phosphate toxicity. Increasing levels of myocardial Pi produced during or immediately following exercise may exacerbate phosphate toxicity associated with SCA in young and older people. Newer technologies are needed to measure changes in cytosolic Pi during myocardial function and explore the novel pathophysiological mechanism proposed in the present paper. Interventions that modify dietary phosphate intake should also be investigated to potentially lower risk of phosphate toxicity associated with SCA.

AUTHOR CONTRIBUTIONS
Ronald B. Brown conceived, researched, designed, and wrote the entire paper.