Renal Failure ISSN: 0886-022X (Print) 1525-6049 (Online) Journal homepage: https://www.tandfonline.com/loi/irnf20 EFFECTS OF HYPOKALEMIA ON CARDIAC GROWTH Zijian Xie, Jiang Liu, Deepak Malhotra, Todd Sheridan, Sankaridrug M. Periyasamy & Joseph I. Shapiro To cite this article: Zijian Xie, Jiang Liu, Deepak Malhotra, Todd Sheridan, Sankaridrug M. Periyasamy & Joseph I. Shapiro (2000) EFFECTS OF HYPOKALEMIA ON CARDIAC GROWTH, Renal Failure, 22:5, 561-572, DOI: 10.1081/JDI-100100897 To link to this article: https://doi.org/10.1081/JDI-100100897 Published online: 07 Jul 2009. Submit your article to this journal Article views: 135 View related articles Citing articles: 15 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=irnf20 RENAL FAILURE, 22(5), 561±572 (2000) LABORATORY STUDY EFFECTS OF HYPOKALEMIA ON CARDIAC GROWTH Zijian Xie, Ph.D., Jiang Liu, Ph.D., Deepak Malhotra, M.D., Ph.D., Todd Sheridan, Sankaridrug M. Periyasamy, Ph.D. and Joseph I. Shapiro, M.D. The Departments of Medicine and Pharmacology Medical College of Ohio ABSTRACT In neonatal myocytes grown in culture, reductions in extracellular potassium concentration produced a hypertrophic response as assessed by induction of early response genes, atrial natriuretic peptide and skeletal actin, and repression of the 3 isoform of the sodium pump in a dose dependent manner. The degree of 3 repression appeared to be dose dependent with decreases in media (K). Similarly, decreases in media potassium concentrations caused increases in cytosolic calcium concentration in a dose dependent manner; moreover these increases in cytosolic calcium concentration correlated quite well with repression of 3 expression. In contrast, although moderate reductions of potassium concentration induced upregulation of skACT and ANP, severely reduced potassium concentrations caused repression of skACT and ANP expression. In parallel studies performed in vivo, 3±5 weeks dietary K restriction induced molecular phenotypical changes similar to that seen in the neonatal myocyte model without demonstrable growth as assessed by the heart weight/body weight ratio. However, when rates subjected Address correspondence to: Joseph I. Shapiro, MD, Chairman, Department of Medicine, Medical College of Ohio, 3120 Glendale Avenue, Toledo, Ohio 43614-5089. Phone: (419) 383-6030; Fax: (419) 383-6244; e-mail: jshapiro@mco.edu 561 Copyright # 2000 by Marcel Dekker, Inc. www.dekker.com 562 XIE ET AL. to dietary K restriction were subsequently subjected to acute aortic constriction, cardiac growth was greater than in rats fed a control diet. These data suggest that hypokalemia may produce molecular phenotypic alterations consistent with cardiac hypertrophy as well as contribute to hypertrophy in an in vivo model. Key Words: Calcium. Sodium; Potassium; ATPase; Hypertrophy; Myocardial; INTRODUCTION Acute and chronic hypokalemia are commonly encountered in a variety of clinical situations (1,2). Of note, physician prescribed acute hypokalemia is standard of care with end stage renal disease patients treated with intermittent hemodialysis (3,4), and hypokalemia frequently complicates diuretic therapy employed in the treatment of hypertension or congestive heart failure (5,6). In addition to having important e€ects on cardiac electrophysiology (7), hypokalemia causes direct impairment of the plasmalemmal sodium pump (8). Recent studies from our laboratory have implicated sodium pump inhibition by cardiac glycosides as a signal transduction pathway operant in the genesis of cardiac cell hypertrophy (9±11). As cardiac hypertrophy frequently develops in the clinical settings listed above, and as this hypertrophy is believed to maladaptive (12±14), we set out to test the hypothesis that sodium pump inhibition induced by hypokalemia might directly cause or facilitate the development of cardiac hypertrophy. METHODS Cell Preparation and Culture Neonatal ventricular myocytes were prepared and cultured as described in previous work from members of our group (9). Brie¯y, myocytes were isolated from ventricles of 1-day-old Sprague-Dawley rats, and puri®ed by centrifugation on Percoll gradients. Myocytes were then cultured in a medium containing 4 parts of DMEM and 1 part Medium 199 (Gibco), penicillin (100 units/mL), streptomycin (100 mg/mL), and 10% fetal bovine serum. After 24 h of incubation at 378C in humidi®ed air with 5% CO2 , medium was changed to one with the same composition as above, but without the serum. All experiments were done after 48 h of further incubation under serum-free conditions. These cultures contain more than 95% HYPOKALEMIA AND CARDIAC GROWTH 563 myocytes as assessed by immuno¯uorescence staining with a myosin heavy chain antibody. Cytosolic Calcium Measurements Cytosolic calcium concentration (Ca) was measured using ¯uorescence spectroscopic imaging utilizing the calcium indicator Fura 2 as described by a number of groups with these cells (15±17). Cells were loaded with 5 uM Fura 2 AM in Krebs Henseleit saline (KHS) containing (Ca) of 1.8 mM and potassium concentration (K) of 5.3 mM at 378C for 15 minutes and then washed multiple times with KHS. During study, cells were perfused with KHS maintained at 378C at 2 mL/min perfusate ¯ow. Because the volume of the perfusion circuit was small, perfusion media (K) could be altered within 15 seconds. An Atto¯or RatioarcTM system (Zeiss Instruments) was employed using excitation wavelengths of 334 and 380 nm and reading ¯uorescence at 510 nm. Cells were studied using a 40X Fluar objective and 340 nm and 380 nm images were obtained using a 0.1 second acquisition time every 15 seconds. Ratio images were constructed from the 340 nm and 380 nm images using Atto¯or software. Approximately 20 voxels were chosen on the ratio image, and the average value was determined for that time point. Ratios were converted to cytosolic calcium using the equation described by Grynkiewicz and colleagues (18). Northern Blot Northern blot was done as previously described (9). Routinely, about 20 ug of total RNA was subjected to gel electrophoresis, transferred to a Nytran membrane, UV-immobilized, and hybridized to 32 P-labeled probes. Autoradiograms obtained at ÿ708C were scanned with a Bio-Rad densitometer. Multiple exposures were analyzed to assure that the signals were within the linear range of the ®lm. The relative amount of mRNA in each sample was normalized to the control value using the GAPDH mRNA amount to correct for di€erences in sample loading and transfer. In Vivo Experiments Male Sprague-Dawley rats (200±250 gm) were fed ad libitum water and experimental low potassium or control diet for 3±4 weeks as we have previously described (19). This diet results in a reduction in serum potassium to approximately 2±2.5 meq/L at this time (20). Rats were then subjected to aortic constriction (produced by tying a silk ligature (4±0) around a 21 gauge 564 XIE ET AL. needle and the suprarenal abdominal aorta and then removing the needle (21±23) or sham surgery and allowed to recover for three days. At his point, the animals were euthanized, the heart weighed and the left ventricle quickly excised and frozen in liquid nitrogen. This frozen tissue was then stored at ÿ808C until it was subsequently analyzed with Northern blotting or enzymatic determination of NaK-ATPase activity using methods described previously (24). Statistical Analysis Data obtained were compared using 1 way analysis of variance (ANOVA) and unpaired Student's t-test with Sche€e's correction for multiple comparisons (25). Statistical analysis was performed using SigmastatTM software. Ethical Considerations All animal experimentation described in the manuscript was conducted in accord with the NIH Guide for the Care and Use of Laboratory Animals using protocols approved by the Medical College of Ohio Institutional Animal Use and Care (IACUC) Committee. RESULTS Neonatal Myocytes Grown in Culture When neonatal myocytes grown in culture were exposed acutely to reductions in potassium concentration, rapid increases in cytosolic (Ca) were observed which transiently stabilized after approximately 5 minutes of perfusion time. Additional mycocyte cultures were subjected to low (K) (2 or 1 mM, both N ˆ 8) for 12 hours and compared to new control coverslips (N ˆ 11). Low potassium concentrations resulted in persistent increases in cytosolic calcium as detailed in Table 1. Neonatal myocytes grown in culture showed signi®cant upregulation of mRNA for ANP and skACT and downregulation of mRNA for 3 NaK-ATPase in a dose dependent fashion in response to prolonged (48 hour) incubation in media containing reduced concentrations of potassium (Table 2). Moderate reductions in extracellular potassium induced ANP and skACT, but a reduction in extracellular (K) to 0.3 mM was associated with decreases in these messages. To further examine this issue, we performed viability studies examining the e€ects of sodium pump inhibition by reduced extracellular potassium on LDH release. Based on these ®ndings summarized in Table 3, it appears that HYPOKALEMIA AND CARDIAC GROWTH 565 Figure 1. Cytosolic calcium concentrations in neonatal myocytes perfused with di€erent potassium concentrations at 5 minutes of experimental perfusion. Data presented as mean SEM of 6±8 measurements under each experimental condition. * p < 0:01 vs Control (K‡) ˆ 5.3 mM. Table 1. E€ect of Reduction in Media (K‡) for 12 Hours on Cytosolic Calcium Determined with Fura 2 Spectroscopic Imaging. Group (N) Control (11) K ˆ 2 mM (8) K ˆ 1 mM (8) (Ca)I (nM) 82 ‡ 7 251 ‡ 44* 358 ‡ 37* Results shown as mean ‡ SEM. * p < 0:01 vs control (K‡) ˆ 5.3 mM. Table 2. E€ect of Reduction in Media (K‡) for 48 Hours on mRNA for Atrial Natriuretic Peptide (ANP), Skeletal Actin (skACT) and the 3 Isoform for NaK-ATPase (3). (K‡) mM 5.3 2.0 1.0 0.3 ANP (% control) skACT (% control) 100 ‡ 5 185 ‡ 20* 245 ‡ 10* 35 ‡ 5* 100 ‡ 4 255 ‡ 15* 442 ‡ 25* 27 ‡ 6* Results shown as mean  SEM. * p < 0:01 vs control (K‡) ˆ 5.3 mM. 3 (% control) 100 ‡ 6 67 ‡ 4* 42 ‡ 2* 9 ‡ 4* 566 XIE ET AL. Table 3. E€ect of Decreased (K‡) on LDH Release. Time (hour) Normalized LDH activity 5.3 5.3 5.3 5.3 0.5 2.0 4.0 16.0 100 99 ‡ 9 99 ‡ 16 100 ‡ 6 2.0 2.0 2.0 2.0 0.5 2.0 4.0 16.0 93 ‡ 8 100 ‡ 19 110 ‡ 7 100 ‡ 5 1.0 1.0 1.0 1.0 0.5 2.0 4.0 16.0 99 ‡ 4 106 ‡ 12 101 ‡ 2 92 ‡ 18 0.3 0.3 0.3 0.3 0.5 2.0 4.0 16.0 101 ‡ 40 229 ‡ 54@ 261 ‡ 46* 245 ‡ 28* (K‡) (mM) Data shown as mean  SEM. @ p < 0:05. * p < 0:01 vs K ˆ 5.33 at 30 min. these cultured cells tolerate media potassium concentrations as low as 1 mM quite well, but that exposure to (K) ˆ 0.3 mM caused signi®cant LDH release. In Vivo Experiments Exposure of rats to 3 weeks of dietary potassium depletion was observed to cause decreases in mRNA for 2 NaK-ATPase and slight induction of mRNA for skACT and ANP (Figure 2, Table 4). In addition, a marked decrease in tissue NaK-ATPase activity was observed with dietary K depletion (Figure 3). However, no growth of such hearts could be observed following 3 weeks of experimental diet as assessed by the heart weight/body weight ratio. The heart weight/body weight ratio is reported rather than the ``raw'' heart weight data because of considerable variability in body weight among the animals. When animals fed the low potassium or control diet were subjected to aortic constriction, marked increases in the heart weight/body weight ratio and decreases in tissue NK-ATPase activity were observed at 3 days p < 0:01† (Figure 3). The increases in heart weight/body weight ratio were greater in the dietary potassium depletion group (Figure 4). Of note, weight loss HYPOKALEMIA AND CARDIAC GROWTH 567 Figure 2. Representative Northern Blot showing mRNA for ANP, skACT and NaKATPase obtained from hearts extracted from normal or hypokalemic animls 3 days following sham or aortic constriction surgery. Lane assignments as follows: ``c'' refers to control diet, ``k'' to low potassium diet, ``0'' refers to no aortic constriction, ``‡'' refers to aortic constriction induced. Table 4. E€ect of Chronic Hypokalemia and Aortic Constriction on mRNA for atrial natriuretic peptide (ANP), skeletal actin (skACT) and the isoform of NaK-ATPase in left ventricular tissue. Group (N) Control (10) Hypokalemia (8) Aortic Constriction (7) Aortic Constriction ‡ Hypokalemia (7) ANP (% control) SkACT (% control) (% control) 100 ‡ 5 152 ‡ 10* 520 ‡ 76* 549 ‡ 48* 100 ‡ 8 119 ‡ 12 352 ‡ 26* 377 ‡ 22* 100 ‡ 3 92 ‡ 12 65 ‡ 5* 55 ‡ 4* Data shown as mean  SEM. * p < 0:01 vs Control. was comparable in the aortic constriction fed the control and low potassium diet 6:4 ‡ 0:5 vs 6:6 ‡ 0:6%, p > 0:20) as well as the control and low potassium diet fed animals subjected to sham surgery 6:3 ‡ 0:7 vs 6:6 ‡ 0:8%, p > 0:20†, and thus, did not obfuscate these ®ndings. Aortic constriction caused marked upregulation of mRNA for skACT and ANP and repression of mRNA for 2 mRNA which were markedly greater in magnitude than that seen from dietary K depletion alone (Figure 2, Table 4). We note that the di€erence in mRNA quantity between hypokalemic and control hearts subjected to 3 days of aortic constriction did not attain statistical signi®cance. 568 XIE ET AL. Figure 3. NaK-ATPase activity in left ventricle of rats subjected to chronic hypokalemia and/or aortic constriction surgery. * p < 0:01 vs control diet & no aortic constriction, # p < 0:01 vs control diet and aortic constriction. Figure 4. Heart weight/Body weight ratios in rats subjected to chronic hypokalemia and/or aortic constriction surgery. * p < 0:01 vs control diet & no aortic constriction, # p < 0:01 vs control diet and aortic constriction. DISCUSSION We have previously demonstrated that acute inhibition of the sodium pump with hypokalemia may induce marked alterations in calcium metabolism in the isolated perfused heart, and that these alterations in calcium HYPOKALEMIA AND CARDIAC GROWTH 569 metabolism may lead to marked functional and metabolic derangements (19,26). Members of our group have also shown that inhibition of the sodium pump with the cardiac glycoside, ouabain reproducibly generates a hypertrophic phenotype in neonatal cardiac myocytes grown in culture through a signaling pathway involving calcium, calmodulin kinase and ras activation (9). This hypertrophic phenotype involves the production of ANP and skACT which are generally regarded as fetal genes and repression of ouabain sensitive chain isoforms of the NaK-ATPase (sodium pump) as recently reviewed (27±30). Based on these points, it seemed reasonable to speculate that sodium pump inhibition with hypokalemia might also produce a hypertrophic phenotype and growth in cardiac myocytes. In our initial studies in neonatal cardiac myocytes grown in culture, hypokalemia produced acute elevations in cytosolic calcium, which were related to the degree of sodium pump inhibition. Based on the observed pseudo Kd of the sodium pump for potassium being 1 mM, one would also predict 20% pump inhibition by decreasing perfusate potassium concentration from 5 to 2 mM (24). Reducing potassium concentration also caused dose dependent reductions in 3 gene expression and increases in skACT and ANP as has previously been observed with ouabain. Although decreases in extracellular potassium concentration to 1 mM were associated with a dose dependent increase in skACT and ANP induction, severe hypokalemia (K) ˆ 0.3 mM) actually repressed skACT and ANP relative to GAPDH message production and impaired cellular viability. In the in vivo experiments, chronic potassium depletion did not by itself cause cardiac hypertrophy although reductions in total cardiac NaK-ATPase activity could be demonstrated. Small changes in 2 skACT and ANP gene expression were associated with the low potassium diet alone. Azuma and coworkers also reported that dietary potassium depletion resulted in comparable decreases in total tissue NaK-ATPase activity to what we report here as well as statistically signi®cant speci®c downregulation of 2 mRNA (31). We observed that the appliction of pressure overload caused marked repression of 2 gene expression and increases in skACT and ANP as well as demonstrable cardiac growth at three days. The addition of the aortic banding to the low potassium diet caused greater hypertrophy than the aortic banding alone although the mRNA concentrations for 2, ANP and skACT were not statistically greater. We would suggest that aortic banding may result in a ``maximal'' repression of 2 and induction of skACT and ANP gene expressions, which cannot be further increased by concomitant potassium depletion despite the augmented growth response seen in this setting. The di€erences between the responses to hypokalemia seen in neonatal cardiac myocytes grown in culture and the in vivo cardiac myocytes may be due to a myriad of factors. One particularly important di€erence is that the young age of the myocytes harvested from the neonatal rats may make them ``primed'' for growth, and therefore more susceptible to stimulation by 570 XIE ET AL. hypokalemia. Also, calcium homeostasis might be substantially di€erent between neonatal cardiac myocytes grown in culture and in vivo cardiac myocytes. In fact, reduction in perfusate K to 2 mM in the isolated heart produced only minimal increases in cytosolic calcium as measured by surface ¯uorescence following Indo-1 loading unless hypoxia was also present (19) in contrast to the data in neonatal cardiac myocytes detailed in this report. 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