Cardiovasc Res 1999 Aug 1;43(2):426-36
Ribadeau-Dumas A, Brady M, Boateng SY, Schwartz K, Boheler KR
 
 

Presenter’s Summary:

Background:

 Myocardial function changes during development, and it is likely that the these changes
occur due to changes in the abundance of sarcoplasmic reticulum (SR) proteins, such as SERCA.
This ATPase facilitates cardiac muscle relaxation by moving Ca2+ from the cytosol to the lumen
of the SR.
 The SERCA proteins act to sequester Ca2+, but three genes (ATP2A1, ATP2A2,
ATP2A3) encode seven protein isoforms, which differ in their regulation (for a review, see
Misquitta et al, 1999).  The SERCA2 gene generates transcripts that are alternatively spliced to
produce a SERCA2a isoform found primarily in cardiac muscle, and  SERCA2b found in
stomach smooth muscle and most other tissues.  This tissue specific splicing results in mRNA
species that differ at their 3' ends and SERCA proteins that differ in their carboxy terminus.  The
proteins are regulated differently, and this may reflect in the different rates of contraction and
relaxation in their native cells.
             Analysis of SERCA2a shows a low affinity for Ca2+ in cardiac muscles due to an
association with the SR membrane protein phospholamban.  Phospholamban is able to inhibit
both SERCA1 and 2 in vitro by lowering their Ca2+ affinity, although phospholamban is
inhibited by phosphorylation.  Ca2+/calmodulin kinase II is able to activate SERCA through the
inhibitory phosphorylation of phospholamban, and directly through SERCA phosphorylation.
 There have been several studies suggesting that SR function is less pronounced in the
fetal heart as compared to the adult rats, and has been linked to an increase in SERCA2a
transcript abundance.  It was previously thought that since SERCA2 gene has a thyroid hormone
responsive promoter sequence that this increase in abundance of mRNA was due to increased
transcription of the gene when thyroid hormone levels increased.
 However, there are numerous ways that a cell can control gene expression, ranging from
the upstream control of transcription itself to the processing of the downstream protein gene
product.   The regulation can be exerted at the level of the DNA, RNA and mature protein, and
consists of a number of complex processes.   One of these processes is the selective degradation
of specific mRNA.
 

Key Findings:

      1.  Using nuclear run ons, in rat cardiac nuclei, no differences could be found in
     SERCA2a transcription from nuclei from rats at 17 embryonic days to nuclei at 20
     days after birth.
      2.  Using dot blots and northern blots, relative to 18S levels and total RNA levels,
     SERCA2a mRNA levels increased after birth with a maximal level of mRNA at
     20 days after birth.
      3.  Using Western Blots and ELISA techniques with a specific anti-SERCA2a
     antibody, there was a significant increase in SERCA2a protein with age.  These
     levels reached a maximum at 20 days after birth.
      4.  By measuring oxalate-supported Ca2+ uptake by the SR in crude homogenates
     during cardiac development, there was a great increase in SERCA activity, but
     plateaued rapidly after birth. The activity prior to birth was 10% of the activity
     even 5 days after birth.
      5.  ELISA techniques demonstrated a significant increase in phospholamban protein
     throughout cardiac development.
 

How is this interpreted?

 The results from the nuclear run-on suggest that there is little difference in the
transcription rates during rat perinatal cardiac development.  This is in opposition to what was
previously suspected.  The interesting finding here, though, is that the mRNA and protein levels
increase while there is no significant increase in mRNA production.  This suggests that there are
post-transcriptional mechanisms regulating this gene’s expression.  It is possible that the mRNA
is stabilized in developing hearts to allow mRNA levels to increase without an increase in
production.
 There are plenty of studies that show that relative stability or instability of mRNA is a
major regulator of gene expression.  Further, another recent study by our group has suggested
that mRNA degradation is a major determinant of SERCA2a and 2b gene expression using
similar methods.  It is suspected that a stable SERCA2a transcript and an unstable 2b transcript
contribute to vastly different mRNA and protein levels in cardiac muscle and stomach smooth
muscle.  This may be a result of the transcripts sequences themselves or various degradative
protein factors native to their tissues.
 In this study, it is possible that native protein factors that change during development
contribute to this increase of mRNA.  There are  no  sequences found in SERCA2a mRNA that
are known to contribute to stability or instability.  The authors do suggest that there may be a
further SERCA2a splice variant that may show differing stability, but levels of these suspected
variants remained in a steady ratio throughout development.  Further studies measuring relative
rates of degradation may be useful to determine whether there is in fact a change in mRNA
stability and how this occurs.
 This study also suggests that post-translational mechanisms are responsible for the
SERCA2a activity changes during development.  This is supported by a coincident increase of
phospholamban with SERCA2a and the plateau of SERCA2a activity.  Incidentally, there was no
change in phosphorylation state of phospholamban during development.
 
 
 

Author’s Abstract:

OBJECTIVE: The Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) plays a major role
in the contraction-relaxation cycle and is responsible for transporting calcium into the lumen of
the sarcoplasmic reticulum. This study was performed to determine if the increase in SERCA2
messenger RNA (mRNA) abundance during the perinatal period is regulated transcriptionally.
METHODS: Transcriptional activity was determined by nuclear run-on assays and mRNA and
protein abundances were determined during late fetal and early neonatal cardiac development in
rat. RESULTS: From nuclear run-on assays, SERCA2 gene transcription at 17/18 embryonic
days (139 +/- 41 parts per million (ppm), n = 7) did not differ from that at 20 neonatal days (139
+/- 37 ppm, n = 6) after birth. No increase in transcriptional activity could be demonstrated
during the time frame examined. In contrast, both alpha and beta myosin heavy chains showed
significant changes in measured transcriptional activity. SERCA2 mRNA normalized to 18S
RNA levels are very low in the fetus (9.8 +/- 1.9 to 13.4 +/- 4.9 arbitrary units (A.U.) from 17/18
to 19/20 embryonic days) and significantly increase from birth (15 +/- 3.8 A.U.) to reach a
maximum at 20 days of age (29.1 +/- 9.5 to 48.3 +/- 7.0 in 15 to 20 neonatal days rats
respectively). Similarly, SR Ca(2+)-ATPase protein levels are less abundant in the fetus (0.82 +/-
0.08 to 1.13 +/- 0.13 A.U./microgram total protein) and reach a maximum at 15-20 neonatal days
(3.08 +/- 0.58 to 2.98 +/- 0.17). Ca2+ uptake in the fetal heart is about one sixth the level seen in
the adult, reaches the highest observed value at 5 days after birth (6.05 +/- 0.77 pmole Ca2+ per
microgram/min) and remains relatively constant over the next 15 days. The activity increases
even though phospholamban protein increases in abundance. CONCLUSIONS: Since the
transcriptional activity of this gene is unchanged whereas the mRNA, protein abundance and
activity increase, we conclude that the abundance of SERCA2 gene products is regulated
primarily through post-transcriptional mechanisms during the perinatal period.