Volume 3, Issue 5, Pages 555-563 (May 1999) Identification of a Specific Role for the Na,K-ATPase α2 Isoform as a Regulator of Calcium in the Heart  Paul F James, Ingrid L Grupp, Gunter Grupp, Alison L Woo, G.Roger Askew, Michelle L Croyle, Richard A Walsh, Jerry B Lingrel  Molecular Cell  Volume 3, Issue 5, Pages 555-563 (May 1999) DOI: 10.1016/S1097-2765(00)80349-4

Figure 1 Strategies for Targeting the Mouse Na,K-ATPase α1 and α2 Isoform Genes (A) Northern blot containing 2 μg of poly(A)-selected RNA (Clonetech) sequentially probed with 32P-labeled alpha isoform–specific oligonucleotide probes demonstrates that only the α1 and the α2 isoforms are expressed in adult mouse heart. (B) Targeting and screening strategy for generation of α1 heterozygous mice. Top, partial structure of the wild-type α1 isoform allele; middle, targeting vector; and bottom, structure of the targeted α1 isoform allele (closed squares denote exons; B denotes BglII sites). (C) Southern blot analysis of genomic DNA from offspring of heterozygous α1 matings; using the probe denoted in (B), a BglII digest of genomic DNA results in a 7.2 kb band for the wild-type allele and a 3.2 kb band for the targeted allele. (D) Targeting and screening strategy for generation of α2 heterozygous mice. Top, partial structure of the wild type α2 isoform allele; middle, targeting vector; and bottom, structure of the targeted α2 isoform allele (RV denotes EcoRV sites). (E) Southern blot analysis of genomic DNA from offspring of heterozygous α2 matings; using the probe denoted in (D), an EcoRV digest of genomic DNA results in a 6.6 kb band for the wild-type allele and a 4.0 kb band for the targeted allele. Molecular Cell 1999 3, 555-563DOI: (10.1016/S1097-2765(00)80349-4)

Figure 2 Analysis of Na,K-ATPase Alpha Isoform mRNA and Protein Levels in Wild-Type, α1, and α2 Heterozygous Hearts (A) Northern blot analysis of poly(A)-selected RNA (5 μg/lane) sequentially probed with 32P-labeled oligonucleotide probes for Na,K-ATPase α1 and α2 isoform and a control (GAPDH) probe. Quantitation of isoform levels from Northern blots containing total RNA (10, 15, and 20 μg) shows that (B) α1 isoform mRNA levels are decreased by ∼40% and α2 isoform mRNA levels are increased by ∼50% in the α1+/− hearts and that (C) α1 isoform mRNA levels are, although variable, similar to wild-type levels and α2 isoform mRNA levels are decreased by ∼50% in the α2+/− hearts. Asterisks denote aberrant transcripts. (D) Western blot analysis of microsomal proteins (40 μg hearts, 10 μg kidney and brain) probed with isoform-specific monoclonal antibodies. Quantitation of isoform protein levels on Western blots containing 10, 20, and 40 μg shows that (E) in the α1+/− hearts, α1 isoform protein levels are decreased by ∼40% and α2 isoform protein levels are increased by ∼50% and that (F) in the α2+/− hearts, α1 isoform protein levels are identical to wild type and α2 isoform protein levels are decreased by ∼40%. Molecular Cell 1999 3, 555-563DOI: (10.1016/S1097-2765(00)80349-4)

Figure 3 Comparison of Contraction and Relaxation Parameters of Isolated Wild-Type, α1+/−, and α2+/− Hearts (A) Representative tracings of, top, intraventricular pressure (IVP) development and, bottom, rates of contraction (+dP/dt) and relaxation (−dP/dt) in wild-type, α1+/−, and α2+/− hearts. (B) Maximal rates of contraction (+dP/dt), (C) maximal rates of relaxation (−dP/dt), (D) time to peak pressure (TPP), and (E) 1/2 relaxation time (RT 1/2) were determined with preload (venous return) fixed at 5 ml/min and afterload (mean aortic pressure) fixed at 50 mm Hg. Heart rates were essentially identical for all three genotypes: wild type, 354 ± 20; α1+/−, 336 ± 10; and α2+/−, 349 ± 20. Wild type, n = 4 hearts; α1+/−, n = 5 hearts; and α2+/−, n = 7 hearts. Note that the α1+/− hearts are hypocontractile; +dP/dt and -dP/dt are reduced, and TPP and RT 1/2 are increased. Also note that the α2+/− hearts are hypercontractile; +dP/dt is increased, and TPP is reduced. Values are reported as mean ± SE. Statistical analysis utilized two-tailed Student’s t test. p values less than 0.05 were considered significant. *p < 0.05; **p < 0.01. Molecular Cell 1999 3, 555-563DOI: (10.1016/S1097-2765(00)80349-4)

Figure 4 Effect of the Cardiac Glycoside Ouabain on Contractility of Wild-Type and α1+/− Hearts (A and B) Representative tracings of, top, intraventricular pressure (IVP) development and, bottom, rates of contraction (+dP/dt) and relaxation (−dP/dt) in (A) wild-type and (B) α1+/− hearts in the presence of 0 M (control), 8 × 10−7 M, and 8 × 10−5 M ouabain. (C) Dose response curve of maximal rate of contraction to increasing concentrations of ouabain. Wild type, n = 4 hearts; α1+/−, n = 5 hearts. Values are reported as mean ± SE. Statistical analysis utilized two-tailed Student’s t test. p values less than 0.05 were considered significant. *p < 0.05 versus α1+/− control; **p < 0.05 versus wild-type control. Molecular Cell 1999 3, 555-563DOI: (10.1016/S1097-2765(00)80349-4)

Figure 5 Functional Compartmentalization Model for Na,K-ATPase Isoform–Specific Functions Colocalization of the Na,K-ATPase α2 isoform and the Na/Ca exchanger to regions of the membrane in close proximity to the sarco/endoplasmic reticulum would provide this Na,K-ATPase isoform specificity in regulating intracellular Ca2+ levels. Inhibition of the Na,K-ATPase α2 isoform would raise the intracellular Na+ concentration in a functional compartment (denoted by dotted line), which would inhibit the Na/Ca exchanger and raise local intracellular Ca2+ concentration. The excess Ca2+ would be transported into the sarcoplasmic reticulum (SR) increasing the SR Ca2+ load, which, when released upon subsequent stimulation, would increase the strength of contractions. Ca2+ Chan., L-type Ca2+ channel; SERCA, sarco/endoplasmic reticulum Ca-ATPase. Molecular Cell 1999 3, 555-563DOI: (10.1016/S1097-2765(00)80349-4)