German Kamalov

Coupled calcium and zinc dyshomeostasis and oxidative stress in cardiac myocytes and mitochondria of rats with chronic aldosteronism.

A dyshomeostasis of extra- and intracellular Ca2+ and Zn2+ occurs in rats receiving chronic aldosterone/salt treatment (ALDOST). Herein, we hypothesized that the dyshomeostasis of intracellular Ca2+ and Zn2+ is intrinsically coupled that alters the redox state of cardiac myocytes and mitochondria, with Ca2+ serving as a pro-oxidant and Zn2+ as an antioxidant. Toward this end, we harvested hearts from rats receiving 4 weeks of ALDOST alone or cotreatment with either spironolactone (Spiro), an aldosterone receptor antagonist, or amlodipine (Amlod), an L-type Ca2+ channel blocker, and from age/sex-matched untreated controls. In each group, we monitored cardiomyocyte [Ca2+]i and [Zn2+]i and mitochondrial [Ca2+]m and [Zn2+]m; biomarkers of oxidative stress and antioxidant defenses; expression of Zn transporters, Zip1 and ZnT-1; metallothionein-1, a Zn2+-binding protein; and metal response element transcription factor-1, a [Zn2+]i sensor and regulator of antioxidant defenses. Compared with controls, at 4-week ALDOST, we found the following: (a) increased [Ca2+]i and [Zn2+]i, together with increased [Ca2+]m and [Zn2+]m, each of which could be prevented by Spiro and attenuated with Amlod; (b) increased levels of 3-nitrotyrosine and 4-hydroxy-2-nonenal in cardiomyocytes, together with increased H2O2 production, malondialdehyde, and oxidized glutathione in mitochondria that were coincident with increased activities of Cu/Zn superoxide dismutase and glutathione peroxidase; and (c) increased expression of metallothionein-1, Zip1 and ZnT-1, and metal response element transcription factor-1, attenuated by Spiro. Thus, an intrinsically coupled dyshomeostasis of intracellular Ca2+ and Zn2+ occurs in cardiac myocytes and mitochondria in rats receiving ALDOST, where it serves to alter their redox state through a respective induction of oxidative stress and generation of antioxidant defenses. The importance of therapeutic strategies that can uncouple these two divalent cations and modulate their ratio in favor of sustained antioxidant defenses is therefore suggested.

Causes and Consequences of Zinc Dyshomeostasis in Rats With Chronic Aldosteronism

Iterations in Ca2+ and Mg2+ balance accompany aldosteronism (inappropriate for dietary Na+ intake). Increased Zn excretion and Zn translocation to injured tissues, including the heart, also occurs. Several causes and consequences of Zn dyshomeostasis in rats receiving aldosterone/salt treatment (ALDOST) were examined. (1) To study the role of urinary acidification in promoting hyperzincuria, acetazolamide (75 mg/kg), a carbonic anhydrase inhibitor, was used as cotreatment to raise urinary HCO3- excretion. (2) To assess Zn levels in the heart, including cardiomyocyte cytosolic free [Zn2+]i and mitochondrial Zn, the expression of metallothionein (MT-I), a Zn binding protein, and biomarkers of oxidative stress were examined. (3) Oxidative stress and cardiac pathology in response to ZnSO4 supplement (40 mg/d) were also studied. Comparison of controls and rats receiving 4 weeks ALDOST revealed the following: (1) an acidification of urine and metabolic alkalosis associated with increased urinary Zn excretion and hypozincemia, each of which were prevented by acetazolamide; (2) a rise in cardiac Zn, including increased [Zn2+]i and mitochondrial Zn, associated with increased tissue MT-I, 8-isoprostane, malondialdehyde, and gp91phox, coupled with oxidative stress in plasma and urine; (3) ZnSO4 prevented hypozincemia, but not ionized hypocalcemia, and attenuated oxidative stress and microscopic scarring without preventing the vasculitis and perivascular fibrosis of intramural coronary arteries. Thus, the hyperzincuria seen with ALDOST is due to urinary acidification. The oxidative stress that appears in the heart is accompanied by increased tissue Zn serving as an antioxidant. Cotreatment with ZnSO4 attenuated cardiomyocyte necrosis; however, polynutrient supplement may be required to counteract the dyshomeostasis of all 3 cations that accompanies aldosteronism and contributes to cardiac pathology.

Temporal responses to intrinsically coupled calcium and zinc dyshomeostasis in cardiac myocytes and mitochondria during aldosteronism

Intracellular Ca(2+) overloading, coupled to induction of oxidative stress, is present at 4-wk aldosterone/salt treatment (ALDOST). This prooxidant reaction in cardiac myocytes and mitochondria accounts for necrotic cell death and subsequent myocardial scarring. It is intrinsically linked to increased intracellular zinc concentration ([Zn(2+)](i)) serving as an antioxidant. Herein, we addressed the temporal responses in coupled Ca(2+) and Zn(2+) dyshomeostasis, reflecting the prooxidant-antioxidant equilibrium, by examining preclinical (week 1) and pathological (week 4) stages of ALDOST to determine whether endogenous antioxidant defenses would be ultimately overwhelmed to account for this delay in cardiac remodeling. We compared responses in cardiomyocyte free [Ca(2+)](i) and [Zn(2+)](i) and mitochondrial total [Ca(2+)](m) and [Zn(2+)](m), together with biomarkers of oxidative stress and antioxidant defenses, during 1- and 4-wk ALDOST. At week 1 and compared with controls, we found: 1) elevations in [Ca(2+)](i) and [Ca(2+)](m) were coupled with [Zn(2+)](i) and [Zn(2+)](m); 2) increased mitochondrial H(2)O(2) production, cardiomyocyte xanthine oxidase activity, and cardiac and mitochondrial 8-isoprostane levels, counterbalanced by increased activity of antioxidant proteins, enzymes, and the nonenzymatic antioxidants that can be considered as cumulative antioxidant capacity; some of these enzymes and proteins (e.g., metallothionein-1, Cu/Zn-superoxide, glutathione synthase) are regulated by metal-responsive transcription factor-1; and 3) although these augmented antioxidant defenses were sustained at week 4, they fell short in combating the persistent intracellular Ca(2+) overloading and marked rise in cardiac tissue 8-isoprostane and mitochondrial transition pore opening. Thus a coupled Ca(2+) and Zn(2+) dyshomeostasis occurs early during ALDOST in cardiac myocytes and mitochondria that regulate redox equilibrium until week 4 when ongoing intracellular Ca(2+) overloading and prooxidants overwhelm antioxidant defenses.

Uncoupling the coupled calcium and zinc dyshomeostasis in cardiac myocytes and mitochondria seen in aldosteronism.

Intracellular [Ca2+]i overloading in cardiomyocytes is a fundamental pathogenic event associated with chronic aldosterone/salt treatment (ALDOST) and accounts for an induction of oxidative stress that leads to necrotic cell death and consequent myocardial scarring. This prooxidant response to Ca2+ overloading in cardiac myocytes and mitochondria is intrinsically coupled to simultaneous increased Zn2+ entry serving as an antioxidant. Herein, we investigated whether Ca2+ and Zn2+ dyshomeostasis and prooxidant to antioxidant dysequilibrium seen at 4 weeks, the pathologic stage of ALDOST, could be uncoupled in favor of antioxidants, using cotreatment with a ZnSO4 supplement; pyrrolidine dithiocarbamate (PDTC), a Zn2+ ionophore; or ZnSO4 in combination with amlodipine (Amlod), a Ca2+ channel blocker. We monitored and compared responses in cardiomyocyte free [Ca2+]i and [Zn2+]i together with biomarkers of oxidative stress in cardiac myocytes and mitochondria. At week 4 of ALDOST and compared with controls, we found (1) an elevation in [Ca2+]i coupled with [Zn2+]i and (2) increased mitochondrial H2O2 production and increased mitochondrial and cardiac 8-isoprostane levels. Cotreatment with the ZnSO4 supplement alone, PDTC, or ZnSO4+Amlod augmented the rise in cardiomyocyte [Zn2+]i beyond that seen with ALDOST alone, whereas attenuating the rise in [Ca2+]i, which together served to reduce oxidative stress. Thus, a coupled dyshomeostasis of intracellular Ca2+ and Zn2+ was demonstrated in cardiac myocytes and mitochondria during 4-week ALDOST, where prooxidants overwhelm antioxidant defenses. This intrinsically coupled Ca2+ and Zn2+ dyshomeostasis could be uncoupled in favor of antioxidant defenses by selectively increasing free [Zn2+]i and/or reducing [Ca2+]i using cotreatment with ZnSO4 or PDTC alone or ZnSO4+Amlod in combination.

Cellular and molecular pathways to myocardial necrosis and replacement fibrosis

Fibrosis is a fundamental component of the adverse structural remodeling of myocardium present in the failing heart. Replacement fibrosis appears at sites of previous cardiomyocyte necrosis to preserve the structural integrity of the myocardium, but not without adverse functional consequences. The extensive nature of this microscopic scarring suggests cardiomyocyte necrosis is widespread and the loss of these contractile elements, combined with fibrous tissue deposition in the form of a stiff in-series and in-parallel elastic elements, contributes to the progressive failure of this normally efficient muscular pump. Cellular and molecular studies into the signal-transducer-effector pathway involved in cardiomyocyte necrosis have identified the crucial pathogenic role of intracellular Ca2+ overloading and subsequent induction of oxidative stress, predominantly confined within its mitochondria, to be followed by the opening of the mitochondrial permeability transition pore that leads to the destruction of these organelles and cells. It is now further recognized that Ca2+ overloading of cardiac myocytes and mitochondria serves as a prooxidant and which is counterbalanced by an intrinsically coupled Zn2+ entry serving as antioxidant. The prospect of raising antioxidant defenses by increasing intracellular Zn2+ with adjuvant nutriceuticals can, therefore, be preferentially exploited to uncouple this intrinsically coupled Ca2+–Zn2+ dyshomeostasis. Hence, novel yet simple cardioprotective strategies may be at hand that deserve to be further explored.

Mitochondria-targeted cardioprotection in aldosteronism

Chronic aldosterone/salt treatment (ALDOST) is accompanied by an adverse structural remodeling of myocardium that includes multiple foci of microscopic scarring representing morphologic footprints of cardiomyocyte necrosis. Our previous studies suggested that signal-transducer-effector pathway leading to necrotic cell death during ALDOST includes intramitochondrial Ca2+ overloading, together with an induction of oxidative stress and opening of the mitochondrial permeability transition pore (mPTP). To further validate this concept, we hypothesized that mitochondria-targeted interventions will prove to be cardioprotective. Accordingly, 8-week-old male Sprague-Dawley rats receiving 4 weeks ALDOST were cotreated with either quercetin, a flavonoid with mitochondrial antioxidant properties, or cyclosporine A (CsA), an mPTP inhibitor, and compared with ALDOST alone or untreated, age/sex-matched controls. We monitored mitochondrial free Ca2+ and biomarkers of oxidative stress, including 8-isoprostane and H2O2 production; mPTP opening; total Ca2+ in cardiac tissue; and collagen volume fraction to quantify replacement fibrosis, a biomarker of cardiomyocyte necrosis, and employed terminal deoxynucleotidyl transferase dUTP nick end labeling assay to address apoptosis in coronal sections of ventricular myocardium. Compared with controls, at 4 weeks ALDOST we found a marked increase in mitochondrial H2O2 production and 8-isoprostane levels, an increased propensity for mPTP opening, and greater concentrations of mitochondrial free [Ca2+]m and total tissue Ca2+, coupled with a 5-fold rise in collagen volume fraction without any terminal deoxynucleotidyl transferase dUTP nick end labeling-based evidence of cardiomyocyte apoptosis. Each of these pathophysiologic responses to ALDOST was prevented by quercetin or cyclosporine A cotreatment. Thus, mitochondria play a central role in initiating the cellular-subcellular mechanisms that lead to necrotic cell death and myocardial scarring. This destructive cycle can be interrupted and myocardium salvaged with its structure preserved by mitochondria-targeted cardioprotective strategies.

From aldosteronism to oxidative stress: the role of excessive intracellular calcium accumulation.

Inappropriately (relative to dietary Na+) elevated plasma aldosterone concentrations (PAC), or aldosteronism, have been incriminated in both the appearance of the cardiometabolic syndrome (CMS) and its progressive nature. The deleterious dual consequences of elevated PAC and dietary Na+ have been linked to several components of the CMS, including salt-sensitive hypertension. Moreover, their adverse consequences are considered to be synergistic, culminating in a pro-oxidant phenotype with oxidative injury involving the heart and systemic tissues, including peripheral blood mononuclear cells (PBMC). Our experimental studies in rats receiving aldosterone/salt treatment have identified a common pathogenic event that links aldosteronism to the induction of oxidative stress. Herein, we review these findings and the important role of excessive intracellular Ca2+ accumulation (EICA), or intracellular Ca2+ overloading, which occurs in the heart and PBMC, leading to, respectively, cardiomyocyte necrosis with a replacement fibrosis and an immunostimulatory state with consequent coronary vasculopathy. The origin of EICA is based on elevations in plasma parathyroid hormone, which are integral to the genesis of secondary hyperparathyroidism that accompanies aldosteronism and occurs in response to plasma-ionized hypocalcemia and hypomagnesemia whose appearance is the consequence of marked urinary and fecal excretory losses of Ca2+ and Mg2+. In addition, we found intracellular Ca2+ overloading to be intrinsically coupled to a dyshomeostasis of intracellular Zn2+, which together regulate the redox state of cardiac myocytes and mitochondria via the induction of oxidative stress and generation of antioxidant defenses, respectively. To validate our hypothesis, a series of site-directed, sequential pharmacological and/or nutriceutical interventions targeted along cellular–molecular cascades were carried out to either block downstream events leading to the pro-oxidant phenotype or to enhance antioxidant defenses. In each case, the interventions were found to be cardioprotective. These cumulative salutary responses raise the prospect that pharmacological agents and nutriceuticals capable of influencing extra- and intracellular Ca2+ and Zn2+ equilibrium could prevent adverse cardiac remodeling and thereby enhance the management of aldosteronism.

Congestive heart failure: where homeostasis begets dyshomeostasis.

Despite todays standard of care, aimed at preventing homeostatic neurohormonal activation, one in every five patients recently hospitalized with congestive heart failure (CHF) will be readmitted within 30 days of discharge because of a recurrence of their symptoms and signs. In light of recent pathophysiological insights, it is now propitious to revisit CHF with a view toward complementary and evolving management strategies. CHF is a progressive systemic illness. Its features include: oxidative stress in diverse tissues; an immunostimulatory state with circulating proinflammatory cytokines; a wasting of soft tissues; and a resorption of bone. Its origins are rooted in homeostatic mechanisms gone awry to beget dyshomeostasis. For example, marked excretory losses of Ca2+ and Mg2+ accompany renin-angiotensin-aldosterone system activation, causing ionized hypocalcemia and hypomagnesemia that lead to secondary hyperparathyroidism with consequent bone resorption and a propensity to atraumatic fractures. Parathyroid hormone accounts for paradoxical intracellular Ca2+ overloading in diverse tissues and consequent systemic induction of oxidative stress. In cardiac myocytes and mitochondria, these events orchestrate opening of the mitochondrial permeability transition pore with an ensuing osmotic-based destruction of these organelles and resultant cardiomyocyte necrosis with myocardial scarring. Contemporaneous with Ca2+ and Mg2+ dyshomeostasis is hypozincemia and hyposelenemia, which compromise metalloenzyme-based antioxidant defenses, whereas hypovitaminosis D threatens Ca2+ stores needed to prevent secondary hyperparathyroidism. An intrinsically coupled dyshomeostasis of intracellular Ca2+ and Zn2+, representing pro-oxidant and antioxidant, respectively, is integral to regulating the mitochondrial redox state; it can be uncoupled by a Zn2+ supplement in favor of antioxidant defenses. Hence, the complementary use of nutriceuticals to nullify dyshomeostatic responses involving macro- and micronutrients should be considered. Evolving strategies with mitochondria-targeted interventions interfering with their uptake of Ca2+ or serving as selective antioxidant or mitochondrial permeability transition pore inhibitor may also prove efficacious in the overall management of CHF.

Gene expression profiles of peripheral blood mononuclear cells reveal transcriptional signatures as novel biomarkers of cardiac remodeling in rats with aldosteronism and hypertensive heart disease.

OBJECTIVES In searching for a noninvasive surrogate tissue having mimicry with the prooxidant/-proinflammatory hypertensive heart disease (HHD) phenotype, we turned to peripheral blood mononuclear cells (PBMC). We tested whether iterations in [Ca2+]i, [Zn2+]i and oxidative stress in cardiomyocytes and PBMC would complement each other eliciting similar shifts in gene expression profiles in these tissues demonstrable during preclinical (wk 1) and pathologic (wk 4) stages of aldosterone/salt treatment (ALDOST). BACKGROUND Inappropriate neurohormonal activation contributes to pathologic remodeling of myocardium in HHD associated with aldosteronism. In rats receiving chronic ALDOST, evidence of reparative fibrosis replacing necrotic cardiomyocytes and coronary vasculopathy appears at wk 4 associated with the induction of oxidative stress by mitochondria that overwhelms endogenous, largely Zn2+-based, antioxidant defenses. Biomarker-guided prediction of risk prior to the appearance of cardiac pathology would prove invaluable. METHODS In PBMC and cardiomyocytes, quantitation of cytoplasmic free Ca2+ and Zn2+, H2O2 and 8-iosprostane levels, as well as isolation of RNA and gene expression, together with statistical and clustering analyses, and confirmation of genes by in situ hybridization and RT-PCR, were performed. RESULTS Compared to controls, at wk 1 and 4 ALDOST, we found comparable: increments in [Ca2+]i, [Zn2+]i and 8-isoprotane coupled to increased H2O2 production in cardiac mitochondria and PBMC, together with the common networks of expression profiles dominated by genes involved in oxidative stress, inflammation and repair. These included three central Ingenuity pathway-linked genes: p38MAPK, a stress-responsive protein; NFκB, a redox-sensitive transcription factor and a proinflammatory cascade it regulates; and TGF-β1, a fibrogenic cytokine involved in tissue repair. CONCLUSIONS Significant overlapping demonstrated in the molecular mimicry of PBMC and cardiomyocytes during preclinical and pathologic stages of ALDOST implicates that transcriptional signatures of PBMC may serve as early noninvasive and novel sentinels predictive of impending pathologic remodeling in HHD.

Atrophic cardiomyocyte signaling in hypertensive heart disease

Abstract: Cardinal pathological features of hypertensive heart disease (HHD) include not only hypertrophied cardiomyocytes and foci of scattered microscopic scarring, a footprint of prior necrosis, but also small myocytes ensnared by fibrillar collagen where disuse atrophy with protein degradation would be predicted. Whether atrophic signaling is concordant with the appearance of HHD and involves oxidative and endoplasmic reticulum (ER) stress remains unexplored. Herein, we examine these possibilities focusing on the left ventricle and cardiomyocytes harvested from hypertensive rats receiving 4 weeks aldosterone/salt treatment (ALDOST) alone or together with ZnSO4, a nonvasoactive antioxidant, with the potential to attenuate atrophy and optimize hypertrophy. Compared with untreated age-/sex-/strain-matched controls, ALDOST was accompanied by (1) left ventricle hypertrophy with preserved systolic function; (2) concordant cardiomyocyte atrophy (<1000 &mgr;m2) found at sites bordering on fibrosis where they were reexpressing &bgr;-myosin heavy chain; and (3) upregulation of ubiquitin ligases, muscle RING-finger protein-1 and atrogin-1, and elevated 8-isoprostane and unfolded protein ER response with messenger RNA upregulation of stress markers. ZnSO4 cotreatment reduced lipid peroxidation, fibrosis, and the number of atrophic myocytes, together with a further increase in cell area and width of atrophied and hypertrophied myocytes, and improved systolic function but did not attenuate elevated blood pressure. We conclude that atrophic signaling, concordant with hypertrophy, occurs in the presence of a reparative fibrosis and induction of oxidative and ER stress at sites of scarring where myocytes are atrophied. ZnSO4 cotreatment in HHD with ALDOST attenuates the number of atrophic myocytes, optimizes size of atrophied and hypertrophied myocytes, and improves systolic function.