ABSTRACT
We have previously reported that genetic reduction of the Na,K-ATPase alpha1 isoform (alpha1(+/-)) results in a hypocontractile cardiac phenotype. This observation was surprising and unexpected. In order to determine if calcium overload contributes to the depressed phenotype, cardiac performance was examined by perfusing the hearts with buffer containing 2 or 1.5 mM calcium. At 2 mM calcium, +dP/dt for the alpha1(+/-) hearts (1374 +/- 180) was significantly less than that of wild-type (2656 +/- 75, P < 0.05). At 1.5 mM calcium, a larger decrease in +dP/dt occurred (vs. 2 mM calcium) for the alpha1(+/-) hearts (517 +/- 92) compared to wild-type (2238 +/- 157). At 2 mM calcium, -dP/dt was 50% lower in alpha1(+/-) hearts (-1903 +/- 141) than wild-type (-982 +/- 143). At 1.5 mM calcium relaxation was further reduced in alpha1(+/-) compared to wild-type (-443 +/- 56 vs. - 1691 +/- 109). We also tested whether the compensatory upregulation of the Na,K-ATPase alpha2 isoform in the alpha1(+/-) hearts contributes to the hypocontractile phenotype. At 8 x 10(-6) M ouabain, that would completely inhibit the alpha2 isoform, a 30% increase in contractility was obtained in alpha1(+/-) hearts compared to no ouabain treatment, while a 63% faster time-to-peak (TTP) and 67% faster half-time-to-relaxation (RT(1/2)) were observed in alpha1(+/-) hearts treated with ouabain. These results suggest that upregulation of the alpha2 isoform may play a role in slower TTP and RT(1/2) in the alpha1(+/-) hearts. Furthermore, lowering extracellular calcium in the perfusate did not alleviate the depressed contractile phenotype in the alpha1(+/-) hearts and resulted in further depressed cardiac contractility suggesting that these hearts are not calcium overloaded.
Subject(s)
Calcium/physiology , Myocardial Contraction/physiology , Sodium-Potassium-Exchanging ATPase/physiology , Animals , Calcium/pharmacology , Mice , Mice, Knockout , Myocardial Contraction/drug effects , Myocardial Contraction/genetics , Myosin Heavy Chains/metabolism , Ouabain/pharmacology , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/biosynthesis , Sodium-Potassium-Exchanging ATPase/deficiency , Sodium-Potassium-Exchanging ATPase/genetics , Tropomyosin/metabolism , Troponin I/metabolism , Up-Regulation , Ventricular Pressure/drug effects , Ventricular Pressure/physiologyABSTRACT
This study compares dihydropyridine receptor (DHPR) and ryanodine receptor (RyR1) gene expression in the diaphragm and hindlimb skeletal muscles of neonatal mice, and examines the contribution of neural and mechanical signals to their developmental induction in vivo. DHPR alpha 1s subunit and RyR1 protein are expressed concurrently, while their respective mRNAs are induced sequentially, with DHPR mRNA ahead of RyR1 mRNA. Both DHPR and RyR1 are more abundant in the diaphragm at birth, and become more abundant in the hindlimb at maturity. These patterns are consistent across different muscles and species. A critical period for DHPR alpha 1s and RyR1 gene expression in the hindlimb occurs between days 5 and 19 postnatal. Their mRNA expression during this period is unchanged by denervation or tenotomy, but DHPR protein decreases after tenotomy. These results demonstrate that both transcriptional and post-transcriptional mechanisms contribute to the muscle-specific and coordinated assembly of the functional DHPR-RyR1 complex, and that the developmental induction of DHPR and RyR1 gene transcription does not require neural or mechanical signals.
Subject(s)
Calcium Channels, L-Type/genetics , Gene Expression Regulation, Developmental/physiology , Neural Pathways/metabolism , Ryanodine Receptor Calcium Release Channel/genetics , Signal Transduction/physiology , Animals , Calcium Channels, L-Type/metabolism , Diaphragm/physiology , Hindlimb/innervation , Hindlimb/physiology , In Vitro Techniques , Mice , Muscle Contraction/physiology , Muscle Denervation , Muscle, Skeletal/physiology , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Ryanodine Receptor Calcium Release Channel/physiology , Signal Transduction/geneticsABSTRACT
This study examined the developmental expression of Na,K-ATPase alpha- and beta-subunit isoforms in different skeletal muscles of the mouse, and the relationship of Na,K-ATPase alpha(2) isoform expression to the developing transverse tubules (t-tubules). We measured Na,K-ATPase and dihydropyridine receptor (DHPR) mRNA and protein in the diaphragm and hindlimb muscles from embryonic day 18.5 (E18.5) to 6 weeks postnatal, using DHPR expression to mark the timing of t-tubule formation. The Na,K-ATPase subunits showed developmental age-dependent and muscle-specific expression that was controlled by both transcriptional and post-transcriptional mechanisms. The alpha(1) isoform is expressed at more constant levels in both diaphragm and hindlimb muscles, while the alpha(2) and beta(2) isoforms increase postnatally and show greater muscle variation. beta(1) is the sole expressed beta-subunit in the diaphragm throughout development, and in the hindlimb muscles at birth. The Na,K-ATPase alpha(2) subunit is expressed during development when the t-tubules form. These results suggest that the alpha(2) isoform may serve, in part, a physiological role in the muscle t-tubules.
