Contractile arrest increases sarcoplasmic reticulum calcium uptake and SERCA2 gene expression in cultured neonatal rat heart cells

José W M Bassani, Ming Qi, Allen M. Samarel, Donald M Bers

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

We developed protocols with intact cultured neonatal rat myocytes to directly evaluate the function of the sarcoplasmic reticulum (SR) Ca-ATPase (or SERCA2), Na-Ca exchange (Na-CaX), and slow Ca transport systems (mitochondria and sarcolemmal Ca-ATPase). Spontaneously heating control cells were compared with cells cultured for 2 days in the presence of verapamil (verapamil-arrested cells VA). Intracellular calcium (Ca(i)) transients were measured by use of indo-1 during (1) spontaneous twitches, (2) contractures induced by rapid application of caffeine (CafC, with and without Na(o)), and (3) twitches induced by brief depolarizations with high [K](o) solution (K- twitches). We also measured mRNA levels for the SR Ca-ATPase and Na-CaX in the same experimental preparations. The t( 1/2 ) for [Ca](i) decline when both the SR Ca uptake and Na-CaX were prevented was the same for control and VA cells (≃20 seconds), indicating unaltered slow Ca transport systems. Similarly, there was no significant difference in the t( 1/2 ) of CafC when Na- CaX was the main mechanism responsible for [Ca](i) decline (t( 1/2 )≃1.5 seconds), indicating unaltered Na-CaX. Conversely, we found nearly a twofold increase in the rate of [Ca](i) decline during K-twitches (control t( 1/2 ), 0.84±0.05 seconds; VA t( 1/2 ), 0.48±0.06 second; P<.001), indicating an increase in SR Ca-pumping activity in VA cells. This was also reflected by a 56% increase in the peak [Ca](i) reached during CafC used to assess maximal SR Ca content (427±49 nmol/L in control versus 665±75 nmol/L in VA cells). In agreement with these functional effects, we found no change in Na-CaX mRNA levels but a marked upregulation of both the SERCA2 mRNA and protein levels in VA cells (to 166±10% and 164±20%, respectively). Thus, verapamil arrest induced an increase in SR Ca uptake (and SERCA2 expression) without affecting the Na-CaX activity (or expression) or the slow Ca transport systems.

Original languageEnglish (US)
Pages (from-to)991-997
Number of pages7
JournalCirculation Research
Volume74
Issue number5
StatePublished - May 1994
Externally publishedYes

Fingerprint

Sarcoplasmic Reticulum
Calcium
Gene Expression
Verapamil
Messenger RNA
Adenosine Triphosphatases
Contracture
Caffeine
Heating
Muscle Cells
Cultured Cells
Mitochondria
Up-Regulation
Proteins

Keywords

  • Ca(i) transients
  • caffeine
  • hypertrophy
  • Na-Ca exchange
  • rat
  • SR Ca-ATPase

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

Contractile arrest increases sarcoplasmic reticulum calcium uptake and SERCA2 gene expression in cultured neonatal rat heart cells. / Bassani, José W M; Qi, Ming; Samarel, Allen M.; Bers, Donald M.

In: Circulation Research, Vol. 74, No. 5, 05.1994, p. 991-997.

Research output: Contribution to journalArticle

@article{f5b6d473c3db4619a93fbfc3d85d7920,
title = "Contractile arrest increases sarcoplasmic reticulum calcium uptake and SERCA2 gene expression in cultured neonatal rat heart cells",
abstract = "We developed protocols with intact cultured neonatal rat myocytes to directly evaluate the function of the sarcoplasmic reticulum (SR) Ca-ATPase (or SERCA2), Na-Ca exchange (Na-CaX), and slow Ca transport systems (mitochondria and sarcolemmal Ca-ATPase). Spontaneously heating control cells were compared with cells cultured for 2 days in the presence of verapamil (verapamil-arrested cells VA). Intracellular calcium (Ca(i)) transients were measured by use of indo-1 during (1) spontaneous twitches, (2) contractures induced by rapid application of caffeine (CafC, with and without Na(o)), and (3) twitches induced by brief depolarizations with high [K](o) solution (K- twitches). We also measured mRNA levels for the SR Ca-ATPase and Na-CaX in the same experimental preparations. The t( 1/2 ) for [Ca](i) decline when both the SR Ca uptake and Na-CaX were prevented was the same for control and VA cells (≃20 seconds), indicating unaltered slow Ca transport systems. Similarly, there was no significant difference in the t( 1/2 ) of CafC when Na- CaX was the main mechanism responsible for [Ca](i) decline (t( 1/2 )≃1.5 seconds), indicating unaltered Na-CaX. Conversely, we found nearly a twofold increase in the rate of [Ca](i) decline during K-twitches (control t( 1/2 ), 0.84±0.05 seconds; VA t( 1/2 ), 0.48±0.06 second; P<.001), indicating an increase in SR Ca-pumping activity in VA cells. This was also reflected by a 56{\%} increase in the peak [Ca](i) reached during CafC used to assess maximal SR Ca content (427±49 nmol/L in control versus 665±75 nmol/L in VA cells). In agreement with these functional effects, we found no change in Na-CaX mRNA levels but a marked upregulation of both the SERCA2 mRNA and protein levels in VA cells (to 166±10{\%} and 164±20{\%}, respectively). Thus, verapamil arrest induced an increase in SR Ca uptake (and SERCA2 expression) without affecting the Na-CaX activity (or expression) or the slow Ca transport systems.",
keywords = "Ca(i) transients, caffeine, hypertrophy, Na-Ca exchange, rat, SR Ca-ATPase",
author = "Bassani, {Jos{\'e} W M} and Ming Qi and Samarel, {Allen M.} and Bers, {Donald M}",
year = "1994",
month = "5",
language = "English (US)",
volume = "74",
pages = "991--997",
journal = "Circulation Research",
issn = "0009-7330",
publisher = "Lippincott Williams and Wilkins",
number = "5",

}

TY - JOUR

T1 - Contractile arrest increases sarcoplasmic reticulum calcium uptake and SERCA2 gene expression in cultured neonatal rat heart cells

AU - Bassani, José W M

AU - Qi, Ming

AU - Samarel, Allen M.

AU - Bers, Donald M

PY - 1994/5

Y1 - 1994/5

N2 - We developed protocols with intact cultured neonatal rat myocytes to directly evaluate the function of the sarcoplasmic reticulum (SR) Ca-ATPase (or SERCA2), Na-Ca exchange (Na-CaX), and slow Ca transport systems (mitochondria and sarcolemmal Ca-ATPase). Spontaneously heating control cells were compared with cells cultured for 2 days in the presence of verapamil (verapamil-arrested cells VA). Intracellular calcium (Ca(i)) transients were measured by use of indo-1 during (1) spontaneous twitches, (2) contractures induced by rapid application of caffeine (CafC, with and without Na(o)), and (3) twitches induced by brief depolarizations with high [K](o) solution (K- twitches). We also measured mRNA levels for the SR Ca-ATPase and Na-CaX in the same experimental preparations. The t( 1/2 ) for [Ca](i) decline when both the SR Ca uptake and Na-CaX were prevented was the same for control and VA cells (≃20 seconds), indicating unaltered slow Ca transport systems. Similarly, there was no significant difference in the t( 1/2 ) of CafC when Na- CaX was the main mechanism responsible for [Ca](i) decline (t( 1/2 )≃1.5 seconds), indicating unaltered Na-CaX. Conversely, we found nearly a twofold increase in the rate of [Ca](i) decline during K-twitches (control t( 1/2 ), 0.84±0.05 seconds; VA t( 1/2 ), 0.48±0.06 second; P<.001), indicating an increase in SR Ca-pumping activity in VA cells. This was also reflected by a 56% increase in the peak [Ca](i) reached during CafC used to assess maximal SR Ca content (427±49 nmol/L in control versus 665±75 nmol/L in VA cells). In agreement with these functional effects, we found no change in Na-CaX mRNA levels but a marked upregulation of both the SERCA2 mRNA and protein levels in VA cells (to 166±10% and 164±20%, respectively). Thus, verapamil arrest induced an increase in SR Ca uptake (and SERCA2 expression) without affecting the Na-CaX activity (or expression) or the slow Ca transport systems.

AB - We developed protocols with intact cultured neonatal rat myocytes to directly evaluate the function of the sarcoplasmic reticulum (SR) Ca-ATPase (or SERCA2), Na-Ca exchange (Na-CaX), and slow Ca transport systems (mitochondria and sarcolemmal Ca-ATPase). Spontaneously heating control cells were compared with cells cultured for 2 days in the presence of verapamil (verapamil-arrested cells VA). Intracellular calcium (Ca(i)) transients were measured by use of indo-1 during (1) spontaneous twitches, (2) contractures induced by rapid application of caffeine (CafC, with and without Na(o)), and (3) twitches induced by brief depolarizations with high [K](o) solution (K- twitches). We also measured mRNA levels for the SR Ca-ATPase and Na-CaX in the same experimental preparations. The t( 1/2 ) for [Ca](i) decline when both the SR Ca uptake and Na-CaX were prevented was the same for control and VA cells (≃20 seconds), indicating unaltered slow Ca transport systems. Similarly, there was no significant difference in the t( 1/2 ) of CafC when Na- CaX was the main mechanism responsible for [Ca](i) decline (t( 1/2 )≃1.5 seconds), indicating unaltered Na-CaX. Conversely, we found nearly a twofold increase in the rate of [Ca](i) decline during K-twitches (control t( 1/2 ), 0.84±0.05 seconds; VA t( 1/2 ), 0.48±0.06 second; P<.001), indicating an increase in SR Ca-pumping activity in VA cells. This was also reflected by a 56% increase in the peak [Ca](i) reached during CafC used to assess maximal SR Ca content (427±49 nmol/L in control versus 665±75 nmol/L in VA cells). In agreement with these functional effects, we found no change in Na-CaX mRNA levels but a marked upregulation of both the SERCA2 mRNA and protein levels in VA cells (to 166±10% and 164±20%, respectively). Thus, verapamil arrest induced an increase in SR Ca uptake (and SERCA2 expression) without affecting the Na-CaX activity (or expression) or the slow Ca transport systems.

KW - Ca(i) transients

KW - caffeine

KW - hypertrophy

KW - Na-Ca exchange

KW - rat

KW - SR Ca-ATPase

UR - http://www.scopus.com/inward/record.url?scp=0028274023&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028274023&partnerID=8YFLogxK

M3 - Article

C2 - 8156646

AN - SCOPUS:0028274023

VL - 74

SP - 991

EP - 997

JO - Circulation Research

JF - Circulation Research

SN - 0009-7330

IS - 5

ER -