Impaired sarcoplasmic reticulum function leads to contractile dysfunction and cardiac hypertrophy.

Sarcoplasmic reticulum (SR)-mediated Ca(2+) sequestration and release are important determinants of cardiac contractility. In end-stage heart failure SR dysfunction has been proposed to contribute to the impaired cardiac performance. In this study we tested the hypothesis that a targeted interference with SR function can be a primary cause of contractile impairment that in turn might alter cardiac gene expression and induce cardiac hypertrophy. To study this we developed a novel animal model in which ryanodine, a substance that alters SR Ca(2+) release, was added to the drinking water of mice. After 1 wk of treatment, in vivo hemodynamic measurements showed a 28% reduction in the maximum speed of contraction (+dP/dt(max)) and a 24% reduction in the maximum speed of relaxation (-dP/dt(max)). The slowing of cardiac relaxation was confirmed in isolated papillary muscles. The late phase of relaxation expressed as the time from 50% to 90% relaxation was prolonged by 22%. After 4 wk of ryanodine administration the animals had developed a significant cardiac hypertrophy that was most prominent in both atria (right atrium +115%, left atrium +100%, right ventricle +23%, and left ventricle +13%). This was accompanied by molecular changes including a threefold increase in atrial natriuretic factor mRNA and a sixfold increase in beta-myosin heavy chain mRNA. Sarcoplasmic endoplasmic reticulum Ca(2+) mRNA was reduced by 18%. These data suggest that selective impairment of SR function in vivo can induce changes in cardiac gene expression and promote cardiac growth.

Phospholamban: a major determinant of the cardiac force-frequency relationship.

The cardiac force-frequency relationship has been known for over a century, yet its mechanisms have eluded thorough understanding. We investigated the hypothesis that phospholamban, a potent regulator of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), determines the cardiac force-frequency relationship. Isolated left ventricular papillary muscles from wild-type (WT) and phospholamban knockout (KO) mice were stimulated at 2 to 6 Hz. The force-frequency relationship was positive in WT but negative in KO muscles, i.e., it was inverted by ablation of phospholamban (P < 0.01, n = 6 mice). From 2 to 6 Hz, relaxation accelerated considerably (by 10 ms) in WT muscles but only minimally (by 2 ms) in KO muscles (WT vs. KO: P < 0. 0001, n = 6). To show that the lack of frequency potentiation in KO muscles was not explained by the almost maximal basal contractility, twitch duration was prolonged in six KO muscles with the SERCA inhibitor cyclopiazonic acid to WT values. Relaxation still failed to accelerate with increased frequency. In conclusion, our results clearly identify phospholamban as a major determinant of the cardiac force-frequency relationship.

Overexpression of sarcoplasmic reticulum Ca(2+)-ATPase improves cardiac contractile function in hypothyroid mice.

OBJECTIVE: Prolonged cardiac contraction and relaxation in hypothyroidism are in part related to diminished expression of the gene coding for the calcium pump of the sarcoplasmic reticulum (SERCA2a). Therefore, we examined whether or not transgenic SERCA2a gene expression in mice may compensate for the cardiac effects of hypothyroidism. METHODS: SERCA2a mRNA and protein were analyzed from hearts of euthyroid and hypothyroid mice of wild-type or SERCA2a transgene status. Contractile function was studied in isolated left ventricular papillary muscles. RESULTS: We found significant decreases of SERCA2a mRNA and protein levels in hearts of hypothyroid wild-type mice in comparison with euthyroid wild-type mice (controls). Papillary muscles from hypothyroid wild-type mice showed significant increases in time to peak contraction and relaxation times compared with controls. In contrast, SERCA2a mRNA and protein levels were significantly higher in hypothyroid SERCA2a transgenic mice than in hypothyroid wild-type mice. The transgene led to a functional improvement by compensating for the prolonged contraction and relaxation of papillary muscles. CONCLUSIONS: Our murine model of hypothyroidism revealed decreases in SERCA2a gene expression accompanied by prolonged contraction and relaxation of papillary muscles, and an improvement of the contractile phenotype due to compensated SERCA2a gene expression in SERCA2a transgenic mice.

Phospholamban-to-SERCA2 ratio controls the force-frequency relationship.

The force-frequency relationship (FFR) describes the frequency-dependent potentiation of cardiac contractility. The interaction of the sarcoplasmic reticulum Ca2+-adenosinetriphosphatase (SERCA2) with its inhibitory protein phospholamban (PLB) might be involved in the control of the FFR. The FFR was analyzed in two systems in which the PLB-to-SERCA2 ratio was modulated. Adult rabbit cardiac myocytes were transduced with adenovirus encoding for SERCA2, PLB, and beta-galactosidase (control). After 3 days, the relative PLB/SERCA2 values were significantly different between groups (SERCA2, 0.5; control, 1.0; PLB, 4.5). SERCA2 overexpression shortened relaxation by 23% relative to control, whereas PLB prolonged relaxation by 39% and reduced contractility by 47% (0.1 Hz). When the stimulation frequency was increased to 1.5 Hz, myocyte contractility was increased by 30% in control myocytes. PLB-overexpressing myocytes showed an augmented positive FFR (+78%), whereas SERCA2-transduced myocytes displayed a negative FFR (-15%). A more negative FFR was also found in papillary muscles from SERCA2 transgenic mice. These findings demonstrate that the ratio of phospholamban to SERCA2 is an important component in the control of the FFR.

Altered cardiac phenotype in transgenic mice carrying the delta337 threonine thyroid hormone receptor beta mutant derived from the S family.

The heart has been recognized as a major target of thyroid hormone action. Our study investigates both the regulation of cardiac-specific genes and contractile behavior of the heart in the presence of a mutant thyroid hormone receptor beta1 (T3Rbeta1-delta337T) derived from the S kindred. The mutant receptor was originally identified in a patient with generalized resistance to thyroid hormone. Cardiac expression of the mutant receptor was achieved by a transgenic approach in mice. As the genes for myosin heavy chains (MHC alpha and MHC beta) and the cardiac sarcoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA2) are known to be regulated by T3, their cardiac expression was analyzed. The messenger RNA levels for MHC alpha and SERCA2 were markedly down-regulated, MHC beta messenger RNA was up-regulated. Although T3 levels were normal in these animals, this pattern of cardiac gene expression mimics a hypothyroid phenotype. Cardiac muscle contraction was significantly prolonged in papillary muscles from transgenic mice. The electrocardiogram of transgenic mice showed a substantial prolongation of the QRS interval. Changes in cardiac gene expression, cardiac muscle contractility, and electrocardiogram are compatible with a hypothyroid cardiac phenotype despite normal T3 levels, indicating a dominant negative effect of the T3Rbeta mutant.

Specific heat shock proteins protect microtubules during simulated ischemia in cardiac myocytes.

The protective effects of heat shock proteins (HSPs) during myocardial ischemia are now well documented, but little is known about the mechanisms of protection and the specificity of different HSPs. Because cytoskeletal injury plays a crucial role in the pathogenesis of irreversible ischemic damage, we tested whether overexpression of specific HSPs protects the integrity of microtubules during simulated ischemia in rat neonatal cardiac myocytes. Overexpression of specific HSPs was achieved by adenovirus-mediated transgene expression. Damage was assessed by comparing control cells to cells that were subjected to a simulated ischemia protocol. Microtubular integrity was measured by indirect immunofluorescence, confocal microscopy, and image analysis. Within 14 h of simulated ischemia, microtubular integrity decreased significantly in uninfected myocytes (from 24.6 +/- 1.2 to 13.2 +/- 0.4) and in myocytes infected with a control virus that expressed no transgene (from 25.9 +/- 1.8 to 13.1 +/- 1.4). Microtubular integrity after ischemia was significantly better preserved in cells overexpressing constitutive Hsp70 (21.7 +/- 1.6) or alphaB-crystallin (18.0 +/- 2.7) but not in cells overexpressing inducible Hsp70 (11.5 +/- 0.8) or Hsp27 (14.0 +/- 2.2). We conclude that specific HSPs protect the microtubules during simulated cardiac ischemia.

Mechanisms of length history-dependent tension in an ionic model of the cardiac myocyte.

The ionic model of the ventricular myocyte developed by Luo and Rudy (Circ. Res. 74: 1071-1096, 1994) was used to investigate potential mechanisms of the slow changes in stress (SCS) that follow step changes in muscle length. A step change in myofilament sensitivity alone caused an immediate increase in active tension, but no SCS. The effects of additional step changes in the parameters of sarcolemmal ion fluxes were examined for each ion flux in the model. Changes in the coefficients of Ca2+ or K+ channels did not produce SCS. SCS was produced by step changes in parameters of the Na(+)-K+ pump or the Na+ leak current. This simulated mechanism was mediated through a slow increase in intracellular Na+ concentration and a resulting increase in systolic Ca2+ entry through the Na+/Ca2+ exchanger. The model reproduced the effects of several experimental interventions such as sarcoplasmic reticulum Ca2+ depletion, "diastolic" length changes, and changes in extracellular Ca2+. Thus SCS in cardiac muscle may be caused by length-induced changes in sarcolemmal Na+ fluxes.

Cellular mechanisms for the slow phase of the Frank-Starling response.

Following a step increase in sarcomere length, isometric cardiac muscle tension increases instantaneously by the Frank-Starling mechanism. In isolated papillary muscle and myocytes, there is an additional significant rise in developed tension over the following 15 min due to an unknown mechanism. This slow change in tension could not be explained by mechanical heterogeneity of the muscle preparations or by an increase in myofilament sensitivity to Ca2+. The slow change in tension was not dependent on sarcoplasmic reticulum Ca2+ loading assessed with rapid cooling contractures, and was not significantly altered by sarcoplasmic reticulum Ca2+ depletion (ryanodine) or inhibition of sarcoplasmic reticulum Ca2+ reuptake (cyclopiazonic acid). We used the Luo-Rudy ionic model of the ventricular myocyte together with a model of the length-dependent myofilament activation by Ca2+ to examine the effects of step changes in the parameters of sarcolemmal ion fluxes as possible mechanisms for the slow change in stress. The slow increase in tension was simulated by step changes in the Na+-K+ pump or Na+ leak currents, suggesting that the slow change in stress may be caused by length induced changes in Na+ fluxes. The model also predicted a slow increase in the magnitude of the initial repolarization during phase 1 of the action potential. The combination of experimental and computational models used in this investigation represents a valuable technique in elucidating the cellular mechanisms of fundamental processes in cardiac excitation-contraction coupling.

Overexpression of the rat sarcoplasmic reticulum Ca2+ ATPase gene in the heart of transgenic mice accelerates calcium transients and cardiac relaxation.

The Ca2+ ATPase of the sarcoplasmic reticulum (SERCA2) plays a dominant role in lowering cytoplasmic calcium levels during cardiac relaxation and reduction of its activity has been linked to delayed diastolic relaxation in hypothyroid and failing hearts. To determine the contractile alterations resulting from increased SERCA2 expression, we generated transgenic mice overexpressing a rat SERCA2 transgene. Characterization of a heterozygous transgenic mouse line (CJ5) showed that the amount of SERCA2 mRNA and protein increased 2. 6-fold and 1.2-fold, respectively, relative to control mice. Determination of the relative synthesis rate of SERCA2 protein showed an 82% increase. The mRNA levels of some of the other genes involved in calcium handling, such as the ryanodine receptor and calsequestrin, remained unchanged, but the mRNA levels of phospholamban and Na+/Ca2+ exchanger increased 1.4-fold and 1.8-fold, respectively. The increase in phospholamban or Na+/Ca2+ exchanger mRNAs did not, however, result in changes in protein levels. Functional analysis of calcium handling and contractile parameters in isolated cardiac myocytes indicated that the intracellular calcium decline (t1/2) and myocyte relengthening (t1/2) were accelerated by 23 and 22%, respectively. In addition, the rate of myocyte shortening was also significantly faster. In isolated papillary muscle from SERCA2 transgenic mice, the time to half maximum postrest potentiation was significantly shorter than in negative littermates. Furthermore, cardiac function measured in vivo, demonstrated significantly accelerated contraction and relaxation in SERCA2 transgenic mice that were further augmented in both groups with isoproterenol administration. Similar results were obtained for the contractile performance of myocytes isolated from a separate line (CJ2) of homozygous SERCA2 transgenic mice. Our findings suggest, for the first time, that increased SERCA2 expression is feasible in vivo and results in enhanced calcium transients, myocardial contractility, and relaxation that may have further therapeutic implications.

Active force in rabbit ventricular myocytes.

Although recent technical advances have established the feasibility of force measurements in single cardiac myocytes, the physiological relevance of this model has not been fully evaluated. We measured active force and sarcomere length in single rabbit left ventricular myocytes and compared their physiological responses to changes in stimulus interval, calcium concentration and sarcomere length to results from isolated papillary muscles. Myocytes were attached to two poly-L-lysine-coated glass plates and force was measured with a capacitive force transducer (Cambridge 406A). Stable recordings from a continuously contracting myocyte could be maintained for over 1 h. In five cells, increasing stimulus interval significantly decreased active force development. This force-stimulus interval relation was similar to that obtained from papillary muscles. In one cell, we obtained a force-length relation that was similar to force-length relations from multicellular preparations. Active stresses (active forces normalized by cross-sectional area) were of similar magnitude when comparing myocytes (at slack length) and papillary muscles (at 85% of Lmax). These results confirm the physiological relevance of force measurements obtained from intact mammalian cardiac myocytes.

Sarcoplasmic reticulum in cardiac length-dependent activation in rabbits.

After a step increase in length of rabbit right ventricular papillary muscles, active stress increased immediately followed by a further slow increase in stress over 15-20 min. We studied the contribution of the sarcoplasmic reticulum (SR) to the slow change in stress (SCS) after changing muscle length from 85 to 95% of length at which active force development was maximal. SCS amounted to 32.5 +/- 12.2% mean +/- SD, n = 19) of the total increase in active stress. This was associated with a 13.2 +/- 8.7% increase in calcium content of the SR as estimated with rapid cooling contractures (P < 0.0001, n = 19). However, SCS was not dependent on SR calcium content. There was no significant attenuation in SCS after SR calcium depletion with ryanodine (n = 6), SR Ca(2+)-adenosinetriphosphatase inhibition with cyclopiazonic acid (n = 6), or combined treatment with ryanodine and cyclopiazonic acid (n = 3). We conclude that, in the rabbit, SR calcium content increases slowly after a step increase in cardiac muscle length but the slow changes in active stress are not dependent on the sarcoplasmic reticulum.