Excitation-contraction coupling in ventricular myocytes is enhanced by paracrine signaling from mesenchymal stem cells.

In clinical trials mesenchymal stem cells (MSCs) are transplanted into cardiac ischemic regions to decrease infarct size and improve contractility. However, the mechanism and time course of MSC-mediated cardioprotection are incompletely understood. We tested the hypothesis that paracrine signaling by MSCs promotes changes in cardiac excitation-contraction (EC) coupling that protects myocytes from cell death and enhances contractility. Isolated mouse ventricular myocytes (VMs) were treated with control tyrode, MSC conditioned-tyrode (ConT) or co-cultured with MSCs. The Ca handling properties of VMs were monitored by laser scanning confocal microscopy and whole cell voltage clamp. ConT superfusion of VMs resulted in a time dependent increase of the Ca transient amplitude (ConT(15min): ΔF/F(0)=3.52±0.38, n=14; Ctrl(15min): ΔF/F(0)=2.41±0.35, n=14) and acceleration of the Ca transient decay (τ: ConT: 269±18ms n=14; vs. Ctrl: 315±57ms, n=14). Voltage clamp recordings confirmed a ConT induced increase in I(Ca,L) (ConT: -5.9±0.5 pA/pF n=11; vs. Ctrl: -4.04±0.3 pA/pF, n=12). The change of τ resulted from increased SERCA activity. Changes in the Ca transient amplitude and τ were prevented by the PI3K inhibitors Wortmannin (100nmol/L) and LY294002 (10µmol/L) and the Akt inhibitor V (20µmol/L) indicating regulation through PI3K signal transduction and Akt activation which was confirmed by western blotting. A change in τ was also prevented in eNOS(-/-) myocytes or by inhibition of eNOS suggesting an NO mediated regulation of SERCA activity. Since paracrine signaling further resulted in increased survival of VMs we propose that the Akt induced change in Ca signaling is also a mechanism by which MSCs mediate an anti-apoptotic effect.

PKC-beta is not necessary for cardiac hypertrophy.

Studies in human and rodent models have shown that activation of protein kinase C-beta (PKC-beta) is associated with the development of pathological hypertrophy, suggesting that ablation of the PKC-beta pathway might prevent or reverse cardiac hypertrophy. To explore this, we studied mice with targeted disruption of the PKC-beta gene (knockout, KO). There were no detectable differences in expression or distribution of other PKC isoforms between the KO and control hearts as determined by Western blot analysis. Baseline hemodynamics were measured using a closed-chest preparation and there were no differences in heart rate and arterial or left ventricular pressure. Mice were subjected to two independent hypertrophic stimuli: phenylephrine (Phe) at 20 mg x kg(-1) x day(-1) sq infusion for 3 days, and aortic banding (AoB) for 7 days. KO animals demonstrated an increase in heart weight-to-body weight ratio (Phe, 4.3 +/- 0.6 to 6.1 +/- 0.4; AoB, 4.0 +/- 0.1 to 5.8 +/- 0.7) as well as ventricular upregulation of atrial natriuretic factor mRNA analogous to those seen in control animals. These results demonstrate that PKC-beta expression is not necessary for the development of cardiac hypertrophy nor does its absence attenuate the hypertrophic response.

Angiotensin II induced cardiac hypertrophy in vivo is inhibited by cyclosporin A in adult rats.

Recently, the calcium-calmodulin-dependent calcineurin pathway has been defined as a central pathway for the induction of cardiac hypertrophy. The purpose of this study was to determine if cardiac hypertrophy in animals chronically treated with angiotensin II (AngII), could be prevented by blocking this pathway with cyclosporin A (CsA). Female Wistar rats were treated with AngII by subcutaneous infusion and injected twice a day with CsA (25 mg/kg) for 7 days. In the AngII treated group there was a 30% increase in the heart/body weight ratio (p < 0.05 vs. control). The increase in heart weight was blocked with CsA. Substantial increases in ANF and betaMHC gene expression were detected in the AngII treated animals, which were either attenuated or blocked with CsA treatment. Thus, this study demonstrates that CsA does prevent the development of cardiac hypertrophy in AngII treated rats, suggesting that the calcium-calmodulin-dependent calcineurin pathway is associated with angiotensin II induced hypertrophy in vivo.

Preservation of glucose metabolism in hypertrophic GLUT4-null hearts.

GLUT4-null mice lacking the insulin-sensitive glucose transporter are not diabetic but do exhibit abnormalities in glucose and lipid metabolism. The most striking morphological consequence of ablating GLUT4 is cardiac hypertrophy. GLUT4-null hearts display characteristics of hypertrophy caused by hypertension. However, GLUT4-null mice have normal blood pressure and maintain a normal cardiac contractile protein profile. Unexpectedly, although they lack the predominant glucose transporter in the heart, GLUT4-null hearts transport glucose and synthesize glycogen at normal levels, but gene expression of rate-limiting enzymes involved in fatty acid oxidation is decreased. The GLUT4-null heart represents a unique model of hypertrophy that may be used to study the consequences of altered substrate utilization in normal and pathophysiological conditions.

beta-adrenergic stimulation causes cardiocyte apoptosis: influence of tachycardia and hypertrophy.

To establish whether catecholamines per se in the absence of significant increases in systolic load induce myocardial damage via apoptosis, rats were treated with vehicle or isoproterenol (400 microg . kg-1 . h-1). Apoptotic cardiocytes (Apo) were identified in paraffin-embedded sections using terminal deoxynucleotide transferase-mediated dUTP nick end labeling. Results were confirmed using an independent ligase assay. Systolic blood pressures were comparable in isoproterenol-treated and control rats. Twenty-four hours of treatment with isoproterenol resulted in significant numbers of Apo compared with control [7.9 +/- 2.5 vs. 0.3 +/- 0.3 (SE) cm-2, P < 0.05]. A cohort of animals was subjected to ventricular pacing to induce a tachycardia equivalent to that induced by isoproterenol, and these animals did not show an increase in Apo. The left ventricular hypertrophy induced by 2 wk of abdominal aortic banding also increased Apo ( approximately 7. 2-fold); however, 24 h of isoproterenol infusion did not induce additional Apo in these rats. Thus catecholamines, in the absence of altered systolic load, induce Apo which is not mediated solely by tachycardia. Left ventricular hypertrophy secondary to abdominal aortic banding is associated with Apo, but this does not increase sensitivity to isoproterenol-induced Apo.

Experimental diabetes is associated with functional activation of protein kinase C epsilon and phosphorylation of troponin I in the heart, which are prevented by angiotensin II receptor blockade.

A cardiomyopathy that is characterized by an impairment in diastolic relaxation and a loss of calcium sensitivity of the isolated myofibril has been described in chronic diabetic animals and humans. To explore a possible role for protein kinase C (PKC)-mediated phosphorylation of myofibrillar proteins in this process, we characterized the subcellular distribution of the major PKC isoforms seen in the adult heart in cardiocytes isolated from diabetic rats and determined patterns of phosphorylation of the major regulatory proteins, including troponin I (TnI). Rats were made diabetic with a single injection of streptozotocin, and myocardiocytes were isolated and studied 3 to 4 weeks later. In nondiabetic animals, 76% of the PKC epsilon isoform was located in the cytosol and 24% was particulate, whereas in diabetic animals, 55% was cytosolic and 45% was particulate (P < .05). PKC delta, the other major PKC isoform seen in adult cardiocytes, did not show a change in subcellular localization. In parallel, TnI phosphorylation was increased 5-fold in cardiocytes isolated from the hearts of diabetic animals relative to control animals (P < .01). The change in PKC epsilon distribution and in TnI phosphorylation in diabetic animals was completely prevented by rendering the animals euglycemic with insulin or by concomitant treatment with a specific angiotensin II type-1 receptor (AT1) antagonist. Since PKC phosphorylation of TnI has been associated with a loss of calcium sensitivity of intact myofibrils, these data suggest that angiotensin II receptor-mediated activation of PKC may play a role in the contractile dysfunction seen in chronic diabetes.

Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates.

Protein kinase C (PKC) activation in the heart has been linked to a hypertrophic phenotype and to processes that influence contractile function. To establish whether PKC activation is sufficient to induce an abnormal phenotype, PKCbeta was conditionally expressed in cardiomyocytes of transgenic mice. Transgene expression in adults caused mild and progressive ventricular hypertrophy associated with impaired diastolic relaxation, whereas expression in newborns caused sudden death associated with marked abnormalities in the regulation of intracellular calcium. Thus, the PKC signaling pathway in cardiocytes has different effects depending on the timing of expression and, in the adult, is sufficient to induce pathologic hypertrophy.

Repeated catecholamine surges alter cardiac isomyosin expression but not protein synthesis in the rat heart.

To assess the role of intermittent beta-adrenergic stimulation on alpha-myosin heavy chain expression and cellular hypertrophy, we studied the effect of intermittent dobutamine on myosin heavy chain isoform distribution and protein synthesis in the heterotopic rat heart preparation. This model allows the analysis of a pharmocologic stimulus in isolation from the mechanical load on the myocardium induced by the drug. Intermittent administration of dobutamine resulted in elevated alpha-MHC levels (75 +/- 12%) compared to control (55 +/- 10%; X +/- s.e.; P<0.05) transplanted hearts. This effect was not altered by alpha-receptor blockade with terazosin (72 +/- 8%). Intermittently pacing the transplanted hearts at the same rate as observed with dobutamine alone, also elevated alpha-MHC levels (70 +/- 5%). In contrast, total protein synthesis in the transplanted hearts was not altered with any of the drug or pacing interventions compared to control hearts. These data suggest that intermittent beta-receptor stimulation and/or intermittent increased heart rate contribute to altered patterns of myosin heavy chain expression. However, increases in cardiac mass and protein synthesis are probably mediated by hemodynamic factors rather than catecholamine stimulation.

GLUT4 heterozygous knockout mice develop muscle insulin resistance and diabetes.

GLUT4, the insulin-responsive glucose transporter, plays an important role in postprandial glucose disposal. Altered GLUT4 activity is suggested to be one of the factors responsible for decreased glucose uptake in muscle and adipose tissue in obesity and diabetes. To assess the effect of GLUT4 expression on whole-body glucose homeostasis, we disrupted the murine GLUT4 gene by homologous recombination. Male mice heterozygous for the mutation (GLUT4 +/-) exhibited a decrease in GLUT4 expression in adipose tissue and skeletal muscle. This decrease in GLUT4 expression did not result in obesity but led to increased serum glucose and insulin, reduced muscle glucose uptake, hypertension, and diabetic histopathologies in the heart and liver similar to those of humans with non-insulin-dependent diabetes mellitus (NIDDM). The male GLUT4 +/- mice represent a good model for studying the development of NIDDM without the complications associated with obesity.

Myocyte apoptosis during acute myocardial infarction in the mouse localizes to hypoxic regions but occurs independently of p53.

Significant numbers of myocytes die by apoptosis during myocardial infarction. The molecular mechanism of this process, however, remains largely unexplored. To facilitate a molecular genetic analysis, we have developed a model of ischemia-induced cardiac myocyte apoptosis in the mouse. Surgical occlusion of the left coronary artery results in apoptosis, as indicated by the presence of nucleosome ladders and in situ DNA strand breaks. Apoptosis occurs mainly in cardiac myocytes, and is shown for the first time to be limited to hypoxic regions during acute infarction. Since hypoxia-induced apoptosis in other cell types is dependent on p53, and p53 is induced by hypoxia in cardiac myocytes, we investigated the necessity of p53 for myocyte apoptosis during myocardial infarction. Myocyte apoptosis occurs as readily, however, in the hearts of mice nullizygous for p53 as in wild-type littermates. These data demonstrate the existence of a p53-independent pathway that mediates myocyte apoptosis during myocardial infarction.

Angiotensin receptor 1 blockade does not prevent physiological cardiac hypertrophy in the adult rat.

The renin-angiotensin system has been implicated in the hypertrophic adaptation of the heart to exogenous pathological loads, such as hypertension and aortic stenosis; however, the role of this hormonal system in the cardiac adaptations to physiological loads, such as chronic exercise conditioning, has not been established. We therefore studied the effect of angiotensin receptor 1 (AT1) blockade on the chronic cardiac responses of rats subjected to an 8-wk swimming program. Compared with matched sedentary controls, untreated swimmers increased their left ventricular weights by 13%, and swimmers treated with the AT1 antagonist L-158809 increased their left ventricular weights by 11% (both P < 0.05 vs. sedentary controls). The incorporation of labeled amino acids into the heart at the time of death was unchanged in all groups, and therefore the increase in heart weight in both swim-conditioned groups appeared to reflect a decrease in the rate of protein degradation in the heart. Hearts from both swim-conditioned groups manifested an increase in the V1-predominant myosin isoform pattern but not an increase in atrial natriuretic factor mRNA expression or protein kinase C translocation. The fact that these patterns of adaptation are preserved in exercised conditioned animals treated with an AT1 antagonist suggests that the chronic hypertrophic response of the heart to physiological loads is not influenced by the renin-angiotensin system.

Ventricular pacing attenuates but does not reverse cardiac atrophy and an isomyosin shift in the rat heart.

The heterotopically transplanted rat heart (TH) undergoes rapid muscle atrophy and a concurrent shift from alpha- to beta-myosin heavy chain (MHC) by 1 wk after surgery. In the current experiments, TH were continuously paced (420 beats/min) for 1 wk beginning 24 h after surgery or for 1 wk beginning 14 days after surgery to determine the role of increased heart rate in preventing or reversing cardiac atrophy. Left ventricular (LV) wet weight (283 vs. 256 mg paced vs. nonpaced) and protein content (32 vs. 23 mg paced vs. nonpaced, P < 0.05) were significantly elevated in TH paced 1 wk after surgery but were unchanged (211 vs. 198 mg and 24 vs. 23 mg LV wet wt and protein content, respectively) in TH paced 2 wk after surgery. Total cardiac protein synthesis in the TH paced immediately after surgery was increased compared with the corresponding nonpaced hearts (5.6 vs. 4.0 mg.mg LV wet wt-1.day-1, P < 0.05), while in the TH, where pacing was initiated 2 wk after surgery, it was unchanged (3.6 vs. 3.7 mg.mg LV wet wt-1.day-1). Fractional synthesis rate was elevated in TH and was not altered by pacing. Pacing the TH also attenuated the shift in alpha-MHC in the first 7 days after surgery but did not reverse the shift 2 wk later. The increase in protein synthesis combined with an unchanged fractional synthesis rate suggests that pacing attenuates cardiac mass by decreasing protein degradation and that once the atrophic process is established, neither synthesis rate nor isomyosin shift can be altered by continuous pacing.

Cardiac adaptations to chronic exercise in mice.

Transgenic mice can be created to serve as models of human cardiac disease. Despite the technology available to manipulate the cardiovascular system of the mouse, there is relatively little information available concerning the normal physiology of the mouse heart. Therefore, we have characterized the response of the adult mouse to chronic physical conditioning by swimming. Adult female C57/B16 mice were conditioned by swimming up to 90 min twice daily for 4 wk, resulting in a 10% increase in heart weight and a 16% increase in heart weight-to-body weight ratios compared with sedentary controls. The heart rate response to a submaximal work load decreased > 20% with this conditioning program. Succinate dehydrogenase activity increased markedly in the soleus muscles of the conditioned animals, from 28 +/- 3 to 44 +/- 3 nmol.mg-1.min-1. In contrast to these changes, which also characterize the exercise model in the rat, no increase in cardiac tissue norepinephrine content or in cardiac myosin or myofibrillar adenosinetriphosphatase (ATPase) activities was observed, and no change in the V1 predominant myosin isoform or alpha-myosin heavy chain mRNA profiles was seen in the hearts of the swimmers. This study establishes that mice are able to develop cardiac hypertrophy in response to chronic conditioning which is not associated with changes in the ATPase activities of cardiac muscle. These data should be of use to investigators using murine models to define the molecular basis of adaptive cardiac hypertrophy in vivo.

Angiotensin II increases cardiac protein synthesis in adult rat heart.

This study examined the direct effect of angiotensin II (ANG II) on cardiac muscle atrophy previously observed in the denervated rat heart. Rats with transplanted hearts were infused with normal saline (1 microliter/h) or a subpressor dose of ANG II dissolved in saline (3 micrograms.kg-1.h-1) for 1 wk. Left ventricular (LV) mass of transplanted hearts decreased by 29 and 18% in the saline-infused and ANG II-infused groups, respectively (P < 0.05). Total LV protein synthesis of the transplanted heart was 1.4 +/- 0.1 mg.LV-1.day-1 in the saline compared with 2.2 +/- 0.2 mg.LV-1.day-1 in the ANG II (P < 0.05) group. Heart rate and carotid systolic arterial pressures were not affected by ANG II infusion, and the decrease in alpha-myosin heavy chain normally observed in this model was unchanged between the two groups (61 +/- 3 and 66 +/- 1%, saline vs. ANG II). These data demonstrate that ANG II increases total cardiac protein synthesis in the adult heart, leading to an attenuation in cardiac atrophy. The failure of ANG II to prevent the shift from alpha- to beta-myosin heavy chain may be related to its lack of an effect on heart rate, since other interventions that affect myosin isoenzyme distribution also increase heart rate.

Increased heart rate prevents the isomyosin shift after cardiac transplantation in the rat.

The heterotopically transplanted rat heart undergoes significant atrophy and a shift from V1 to V3 isomyosin. The purpose of this study was to pace the cardiac isograft and determine whether an increase in heart rate would attenuate the changes in cardiac mass and isoenzyme distribution. Nonpaced transplanted hearts were compared with hearts in which pacing was initiated at 7 Hz, 24 hours after transplantation, and continued for 7 days. There was a 29% decrease in myosin ATPase activity and a 22% decrease in alpha-myosin in the nonpaced isograft; both decreases were completely prevented by pacing. The decrease in cardiac mass was also significantly attenuated. Pacing did not alter intrinsic heart rate, systolic pressure, dP/dt, or norepinephrine concentration in the isograft. These results suggest that the adaptation in both cardiac mass and isoenzymes may be related to the rate or the rate-pressure product in the transplanted paced heart independent of left ventricular pressure, tissue catecholamines, or neural activity.

Ventricular function and contractile proteins in the infarcted overloaded rat heart.

STUDY OBJECTIVE: The aim was to determine whether surviving myocardium in the infarcted rat heart retains the ability to respond to sustained increases in afterload. DESIGN: Cardiac mass, ventricular function, and actomyosin ATPase activity were compared in animals subjected to coronary artery ligation to produce infarction, superimposed renal artery constriction 4 weeks after infarction, and in sham operated animals. EXPERIMENTAL MATERIAL: Female Wistar rats obtained at 10 weeks of age (200-225 g) were used for the studies. MEASUREMENTS AND MAIN RESULTS: Four weeks after coronary artery ligation, infarcted hearts showed a 22% increase in heart weight and a significant reduction in peak systolic pressure and +/- dP/dt during acute volume infusion and aortic occlusion compared to sham operated hearts. Eight weeks after the initial surgical intervention, the infarct group showed significant impairment in ventricular performance compared to the sham operated group but no further decrement was observed between hearts with infarction and those with infarct and superimposed renal artery constriction for peak systolic pressure and +/- dP/dt during volume infusion and aortic occlusion. Actomyosin ATPase activity, however, was depressed and the shift to V3 myosin isoenzyme was greater in infarct and renal artery constriction compared to infarct alone. CONCLUSIONS: Left ventricular myocardium following infarction does not retain the ability to increase cardiac mass and shows depressed levels of actomyosin ATPase activity when exposed to a superimposed chronic afterload from renal artery constriction. However, cardiac function in situ is maintained.

Regional variation in rat cardiac myosin isoenzymes and ATPase activity after infarction.

Three and 11 wk after coronary artery ligation in rats, the right and left ventricular free wall, septum, and papillary muscles from infarcted and sham-operated hearts were analyzed to determine whether regional variability existed in cardiac actomyosin adenosine triphosphate (ATPase) activity and myosin isoenzymes. Infarction produced a 74% greater right ventricular mass and 19% greater septal mass compared with sham-operated hearts at 3 wk. There was no additional increase in cardiac mass associated with infarction from 3 to 11 wk above that expected for normal growth. Actomyosin ATPase activity and the percent V1 myosin heavy-chain isoenzyme decreased significantly in all regions of the infarcted heart by 3 wk. In addition, the left ventricular and papillary muscle of infarcted hearts exhibited a decrease in percent V1 myosin of 18 and 35%, respectively, compared with the right ventricular free wall and septum. These differences persisted at 11 wk, although no further depression of actomyosin ATPase activity or shift in myosin isoenzyme distribution were observed over the 8-wk period. These results demonstrate that myocardial infarction induces a shift in the myosin isoenzyme distribution and depression in actomyosin ATPase activity of surviving cardiac tissue. Regional variability in myosin isoenzymes is evident by 3 wk, but additional adaptation in cardiac mass and myosin biochemistry do not occur beyond this time.

Papillary mechanics and cardiac morphology of infarcted rat hearts after training.

Infarction of the left ventricle was induced by ligation of the coronary artery in male Sprague-Dawley rats under ketamine-xylazine anesthesia. Three weeks after surgery, animals were assigned to a trained (n = 21; running at 20 m/min, 10% grade, 1 h/day, 5 days/wk) or nontrained group (n = 23) for an additional 8 wk. A third, sham-operated control group (n = 16) remained cage sedentary for 11 wk. Ventricular mass was greater in the trained and nontrained infarct groups [1,335 +/- 57.3 and 1,414 +/- 56.1 mg, respectively (mean +/- SE)] compared with the control group (1,155 +/- 50.9 mg) (P less than or equal to 0.05). The diameter of septal fibers was 13% greater in the trained and 17% greater in the nontrained infarct groups compared with control. The specific peak developed force and maximum rate of force development of left ventricular papillary muscle in vitro were 75 and 62% greater in both infarcted groups compared with the control group; these variables were unaffected by training. Myofibrillar adenosine triphosphatase activity of septum was 20% lower in both infarct groups compared with sham-operated animals. We conclude that exercise training did not alter the magnitude of morphological and physiological adaptations to infarction.

Daily physical activity patterns of prepubertal children involved in a vigorous exercise program.

The purpose of the present investigation was to examine the effects of a vigorous physical activity program on daily physical activity patterns of 59 7-year-old children divided into experimental (n = 26) and control (n = 33) groups. The experimental group participated in a 25-min vigorous, aerobic exercise session 4 days per week, while the control group maintained their normal daily activities, which included a 1 day per week physical education class, for 8 months. The intensity of each experimental exercise session and each control physical education class was determined by fitting Exersentry heart rate devices to two different children randomly selected from each group without replacement. Daily activity patterns were determined using minute-by-minute heart rates calculated from a 12-h EKG recorded from 8 a.m. to 8 p.m. using an Oxford Instruments ambulatory monitor. Analysis of the Exersentry data demonstrated significantly higher heart rates (P less than 0.05) during the experimental exercise session compared to the control physical education class, except during pre-exercise. Analysis of the 12-h EKG data (n = 720 min) revealed the experimental group spent significantly more time (P less than 0.05) at heart rates greater than 160 bts X min-1 (35 +/- 6 min, experimental; 21 +/- 3 min, control) during the intervention program. No significant group differences (P greater than 0.05) were observed in 12-h heart rate data collected prior to initiation of the intervention program. These data suggest that a vigorous physical activity program resulted in differences in the daily physical activity patterns of 7-year-old children.