Pregnancy late in rodent life has detrimental effects on the heart.

During pregnancy, the heart undergoes significant and numerous changes, including hypertrophy, that are usually described as physiological and reversible. Two aspects of the cardiac response to pregnancy are relatively understudied: advanced maternal age and multiple pregnancies (multiparity). Repeated breeder (RB) mice that have undergone five to seven consecutive pregnancies were euthanized 21 days after the weaning of their last pups and compared with age-matched primiparous, one-time pregnant (O1P) mice. The ages of the older mouse groups were similar (12 ± 1 mo). Pregnancy at a later age resulted in reduced fertility (40%); resorption was 29%, maternal mortality was 10%, and mortality of the pups was 17%. Contractile function as indicated by percent fractional shortening was significantly decreased in O1P and RB groups compared with the old nonpregnant control (ONP) group. There was no pathological induction of the fetal program of gene expression, with the exception of ß-myosin heavy chain mRNA, which was induced in O1P compared with ONP mice ( P < 0.05) but not in RB mice. MicroRNA-208a was significantly increased in O1P compared with ONP mice ( P < 0.05) but significantly decreased in RB compared with ONP mice ( P < 0.05). mRNA of genes regulating angiogenesis (i.e., vascular endothelial growth factor-A) were significantly downregulated, whereas proinflammatory genes [i.e., interleukin-6, chemokine (C-C motif) ligand 2, and Cd36] were significantly upregulated in O1P ( P < 0.05) but not in RB mice. Overall, our results suggest that rather than multiparity, pregnancy in advanced age is a much more stressful event in both pregnant dams and fetuses, as evidenced by increased mortality, lower fertility, downregulation of angiogenesis, upregulation of inflammation, and cardiac dysfunction. NEW & NOTEWORTHY Pregnancy in older mice significantly decreases cardiac function, although repeated breeder mice demonstrated increased wall hypertrophy and dilated chamber size compared with one-time pregnant mice. Interestingly, many of the molecular changes were altered in one-time pregnant mice but not in repeated breeder mice, which may contribute to adverse pregnancy outcomes in a first pregnancy at a later age.

Effect of exercise training and myocardial infarction on force development and contractile kinetics in isolated canine myocardium.

It is well known that moderate exercise training elicits a small increase in ventricular mass (i.e., a physiological hypertrophy) that has many beneficial effects on overall cardiac health. It is also well known that, when a myocardial infarction damages part of the heart, the remaining myocardium remodels to compensate for the loss of viable functioning myocardium. The effects of exercise training, myocardial infarction (MI), and their interaction on the contractile performance of the myocardium itself remain largely to be determined. The present study investigated the contractile properties and kinetics of right ventricular myocardium isolated from sedentary and exercise trained (10-12 wk progressively increasing treadmill running, begun 4 wk after MI induction) dogs with and without a left ventricular myocardial infarction. Exercise training increased force development, whereas MI decreased force development that was not improved by exercise training. Contractile kinetics were significantly slower in the trained dogs, whereas this impact of training was less or no longer present after MI. Length-dependent activation, both evaluated on contractile force and kinetics, was similar in all four groups. The control exercise-trained group exhibited a more positive force-frequency relationship compared with the sedentary control group while both sedentary and trained post-MI dogs had a more negative relationship. Last, the impact of the ß-adrenergic receptor agonist isoproterenol resulted in a similar increase in force and acceleration of contractile kinetics in all groups. Thus, exercise training increased developed force but slowed contractile kinetics in control (noninfarcted animals), actions that were attenuated or completely absent in post-MI dogs.

Dissociation of Calcium Transients and Force Development following a Change in Stimulation Frequency in Isolated Rabbit Myocardium.

As the heart transitions from one exercise intensity to another, changes in cardiac output occur, which are modulated by alterations in force development and calcium handling. Although the steady-state force-calcium relationship at various heart rates is well investigated, regulation of these processes during transitions in heart rate is poorly understood. In isolated right ventricular muscle preparations from the rabbit, we investigated the beat-to-beat alterations in force and calcium during the transition from one stimulation frequency to another, using contractile assessments and confocal microscopy. We show that a change in steady-state conditions occurs in multiple phases: a rapid phase, which is characterized by a fast change in force production mirrored by a change in calcium transient amplitude, and a slow phase, which follows the rapid phase and occurs as the muscle proceeds to stabilize at the new frequency. This second/late phase is characterized by a quantitative dissociation between the calcium transient amplitude and developed force. Twitch timing kinetics, such as time to peak tension and 50% relaxation rate, reached steady-state well before force development and calcium transient amplitude. The dynamic relationship between force and calcium upon a switch in stimulation frequency unveils the dynamic involvement of myofilament-based properties in frequency-dependent activation.

Post-translational modifications of myofilament proteins involved in length-dependent prolongation of relaxation in rabbit right ventricular myocardium.

The phosphorylation state of several cardiac myofilament proteins changes with the level of stretch in intact, twitch-contracting cardiac muscles. It remains unclear which kinases are involved in the length-dependent phosphorylation of these proteins. We set out to investigate which kinases are involved after a step-wise change in cardiac muscle length. We hypothesize that myofilament protein phosphorylation by PKCßII and PKA alters contractile kinetics during length-dependent activation. Right ventricular intact trabeculae were isolated from New Zealand White rabbit hearts and stimulated to contract at 1Hz. Twitch force recordings where taken at taut and optimal muscle lengths before and after administration of kinase inhibitors at 37°C. PKCßII inhibition significantly decreased time from stimulation to peak force (TTP), time from peak force to 50% relaxation (RT50), and 90% relaxation (RT90) at optimal muscle length. This led to a loss in the length-dependent increase of RT50 and RT90 in the presence of the PKCßII inhibitor, whereas the length-dependent increase in RT50 and RT90 was seen in the controls. PKA inhibition using H-89 significantly decreased TTP at both taut and optimal muscle lengths. Detection of Ser/Thr phosphorylation with ProQ-diamond staining indicates a role for PKCßII in the phosphorylation of tropomyosin and myosin light chain-2 (MLC2) and PKA for tropomyosin, troponin-I, MLC2, myosin binding protein-C, troponin-T (TnT) 3 and TnT4. Our data provide evidence for two signaling kinases acting upon myofilament proteins during length-dependent activation, and provide further insight for length-dependent myofilament function.

Role of endothelin in the induction of cardiac hypertrophy in vitro.

Endothelin (ET-1) is a peptide hormone mediating a wide variety of biological processes and is associated with development of cardiac dysfunction. Generally, ET-1 is regarded as a molecular marker released only in correlation with the observation of a hypertrophic response or in conjunction with other hypertrophic stress. Although the cardiac hypertrophic effect of ET-1 is demonstrated, inotropic properties of cardiac muscle during chronic ET-1-induced hypertrophy remain largely unclear. Through the use of a novel in vitro multicellular culture system, changes in contractile force and kinetics of rabbit cardiac trabeculae in response to 1 nM ET-1 for 24 hours can be observed. Compared to the initial force at t = 0 hours, ET-1 treated muscles showed a ~2.5 fold increase in developed force after 24 hours without any effect on time to peak contraction or time to 90% relaxation. ET-1 increased muscle diameter by 12.5 ± 3.2% from the initial size, due to increased cell width compared to non-ET-1 treated muscles. Using specific signaling antagonists, inhibition of NCX, CaMKII, MAPKK, and IP3 could attenuate the effect of ET-1 on increased developed force. However, among these inhibitions only IP3 receptor blocker could not prevent the increase muscle size by ET-1. Interestingly, though calcineurin-NFAT inhibition could not suppress the effect of ET-1 on force development, it did prevent muscle hypertrophy. These findings suggest that ET-1 provokes both inotropic and hypertrophic activations on myocardium in which both activations share the same signaling pathway through MAPK and CaMKII in associated with NCX activity.

Impact of hydroxyl radical-induced injury on calcium handling and myofilament sensitivity in isolated myocardium.

Hydroxyl radicals (OH) are involved in the pathogenesis of reperfusion injury and are observed in acute heart failure, stroke, and myocardial infarction. Two different subcellular defects are involved in the pathogenesis of OH injury, deranged calcium handling, and alterations of myofilament responsiveness, but their temporal impact on contractile function is not resolved. Initially, after brief OH exposure, there is a corresponding marked increase in diastolic calcium and diastolic force. We followed these parameters until a new steady-state level was reached at ~45 min post-OH exposure. At this new baseline, diastolic calcium had returned to near-normal, pre-OH levels, whereas diastolic force remained markedly elevated. An increased calcium sensitivity was observed at the new baseline after OH-induced injury compared with the pre-OH state. The acute injury that occurs after OH exposure is mainly due to calcium overload, while the later sustained myocardial dysfunction is mainly due to the altered/increased myofilament responsiveness.

Effects of increased preload on the force-frequency response and contractile kinetics in early stages of cardiac muscle hypertrophy.

Numerous studies have aimed to elucidate markers for the onset of decompensatory hypertrophy and heart failure in vivo and in vitro. Alterations in the force-frequency relationship are commonly used as markers for heart failure with a negative staircase being a hallmark of decompensated cardiac function. Here we aim to determine the functional and molecular alterations in the very early stages of compensatory hypertrophy through analysis of the force-frequency relationship, using a novel isolated muscle culture system that allows assessment of force-frequency relationship during the development of hypertrophy. New Zealand white male rabbit trabeculae excised from the right ventricular free wall were utilized for all experiments. Briefly, muscles held at constant preload and contracting isometrically were stimulated to contract in culture for 24 h, and in a subset up to 48 h. We found that, upon an increase in the preload and maintaining the muscles in culture for up to 24 h, there was an increase in baseline force produced by isolated trabeculae over time. This suggests a gradual compensatory response to the impact of increased preload. Temporal analysis of the force-frequency response during this progression revealed a significant blunting (at 12 h) and then reversal of the positive staircase as culture time increased (at 24 h). Phosphorylation analysis revealed a significant decrease in desmin and troponin (Tn)I phosphorylation from 12 to 24 h in culture. These results show that even very early on in the compensatory hypertrophy state, the force-frequency relationship is already affected. This effect on force-frequency relationship may, in addition to protein expression changes, be partially attributed to the alterations in myofilament protein phosphorylation.

Effect of twitch interval duration on the contractile function of subsequent twitches in isolated rat, rabbit, and dog myocardium under physiological conditions.

Many studies have shown that a change in stimulation frequency leads to altered contractility of the myocardium. However, it remains unclear what changes occur directly after a change in frequency and which ones are a result of the slow processes that lead to the altered homeostasis, which develops after a change in stimulation frequency. To distinguish the immediate from the slow responses, we assessed contractile function in two species that have distinctively different calcium (Ca(2+))-handling properties using a recently developed, randomized pacing protocol. In isolated dog and rat right ventricular trabeculae, twitch contractions at five different cycle lengths within the physiologic range of each species were randomized around a steady-state frequency. We found, in both species, that the duration of the cycle length just prior to the analyzed twitch (primary) positively correlated with the increased force of the analyzed twitch. In sharp contrast, the cycle lengths, one and two more removed from the analyzed twitch ("secondary" and "tertiary"), displayed a negative correlation with force of the analyzed twitch. In additional experiments, assessment of intracellular Ca(2+) transients in rabbit trabeculae revealed that diastolic Ca(2+) levels were closely correlated to contractile function outcome. The relative contribution of the primary cycle length was different between dog (51%) and rat (71%), whereas in neither species was a significant effect on relaxation time observed. With the use of randomized cycle lengths, we have distinguished the intrinsic response from the signaling-mediated effects of frequency-dependent activation on myofilament properties and Ca(2+) handling.

In vitro studies of early cardiac remodeling: impact on contraction and calcium handling.

Cardiac remodeling, hypertrophy, and alterations in calcium signaling are changes of the heart that often lead to failure. After a hypertrophic stimulus, the heart progresses through a state of compensated hypertrophy which over time leads to decompensated hypertrophy or failure. It is at this point that a cardiac transplant is required for survival making early detection imperative. Current experimental systems used to study the remodeling of the heart include in vivo systems (the whole body), isolated organ and sub-organ tissue, and the individual cardiac muscle cells and organelles.. During pathological remodeling there is a derangement in the intracellular calcium handling processes. These derangements are thought to lead to a dysregulation of contractile output. Hence, understanding the mechanism between remodeling and dysregulation is of great interest in the cardiac field and will ultimately help in the development of future treatment and early detection. This review will center on changes in contraction and calcium handling in early cardiac remodeling, with a specific focus on findings in two different in vitro model systems: multicellular and individual cell preparations.

Effects of dietary omega-3 fatty acids on ventricular function in dogs with healed myocardial infarctions: in vivo and in vitro studies.

Since omega-3 polyunsaturated fatty acids (n-3 PUFAs) can alter ventricular myocyte calcium handling, these fatty acids could adversely affect cardiac contractile function, particularly following myocardial infarction. Therefore, 4 wk after myocardial infarction, dogs were randomly assigned to either placebo (corn oil, 1 g/day, n = 16) or n-3 PUFAs supplement [docosahexaenoic acid (DHA) + eicosapentaenoic acid (EPA) ethyl esters; 1, 2, or 4 g/day; n = 7, 8, and 12, respectively] groups. In vivo, ventricular function was evaluated by echocardiography before and after 3 mo of treatment. At the end of the 3-mo period, hearts were removed and in vitro function was evaluated using right ventricular trabeculae and isolated left ventricular myocytes. The treatment elicited significant (P < 0.0001) dose-dependent increases (16.4-fold increase with 4 g/day) in left ventricular tissue and red blood cell n-3 PUFA levels (EPA + DHA, placebo, 0.42 +/- 0.04; 1 g/day, 3.02 +/- 0.23; 2 g/day, 3.63 +/- 0.17; and 4 g/day, 6.97 +/- 0.33%). Regardless of the dose, n-3 PUFA treatment did not alter ventricular function in the intact animal (e.g., 4 g/day, fractional shortening: pre, 42.9 +/- 1.6 vs. post, 40.1 +/- 1.7%; placebo: pre, 39.2 +/- 1.3 vs. post, 38.4 +/- 1.6%). The developed force per cross-sectional area, changes in length- and frequency-dependent behavior in contractile force, and the inotropic response to beta-adrenoceptor activation were also similar for trabeculae obtained from placebo- or n-3 PUFA-treated dogs. Finally, calcium currents and calcium transients were the same in myocytes from n-3 PUFA- and placebo-treated dogs. Thus dietary n-3 PUFAs did not adversely alter either in vitro or in vivo ventricular contractile function in dogs with healed infarctions.

Temporal changes in expression of connexin 43 after load-induced hypertrophy in vitro.

Upon remodeling of the ventricle after a provoking stimulus, such as hypertension, connections between adjacent myocytes may need to be "reformatted" to preserve a synchronization of excitation of the remodeling heart. In the mammalian heart, the protein connexin forms the gap junctions that allow electrical and chemical signaling communication between neighboring cells. We aim to elucidate whether mechanical load, in isolation, potentially changes the expression of connexin 43 (Cx43), the major isoform of the connexin family in the ventricle, and its phosphorylation. Cx43 expression levels and contractile function of multicellular rabbit cardiac preparations were assessed in a newly developed in vitro system that allows for the study of the transition of healthy multicellular rabbit myocardium to hypertrophied myocardium. We found that in mechanically loaded cardiac trabeculae, Cx43 levels remained stable for about 12 h and then rapidly declined. Phosphorylation at Ser368 declined much faster, being almost absent after 2 h of high-load conditions. No-load conditions did not affect Cx43 levels, nor did phosphorylation at Ser368. The downregulation of Cx43 under mechanical load did not correspond with the contractile changes that were observed. Furthermore, blocking paracrine activity of the muscle could only partially prevent the downregulation of Cx43. Additionally, no effect of mechanical loading on the expression of N-cadherin and zonula occludens-1 was observed, indicating a specificity of the connexin response. High mechanical load induced a rapid loss of Cx43 phosphorylation, followed by a decrease in Cx43 protein levels. Paracrine factors are partly responsible for the underlying mechanism of action, whereas no direct correlation to contractile ability was observed.