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.

Mouse model of a familial hypertrophic cardiomyopathy mutation in alpha-tropomyosin manifests cardiac dysfunction.

To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant alpha-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the alpha-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous alpha-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain alpha-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca2+ sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC alpha-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.

A4 amyloid protein deposition and the diagnosis of Alzheimer's disease: prevalence in aged brains determined by immunocytochemistry compared with conventional neuropathologic techniques.

The histologic diagnosis of Alzheimer's disease (AD) might be aided if a more sensitive marker of aberrant A4 amyloid protein deposition were available. We screened a sample of aged brains, using immunocytochemical methods to detect the A4 protein deposition, and found that, in comparison with conventional histologic techniques (silver impregnation and Congo red), immunocytochemistry is more sensitive and allows an easier demarcation between "normal" and "abnormal." If A4 protein deposition is accepted as a definitive marker for AD, then the age-related prevalence of AD increases dramatically. To what degree these prevalence rates are reflected in clinically detectable impairment of higher cortical function remains to be determined.

The effect of thapsigargin on sarcoplasmic reticulum Ca2+ content and contractions of single myocytes of rat ventricular myocardium.

The effect of Thapsigargin (TG) shown to block selectively the Ca(2+)-ATPase of sarcoplasmic reticulum (SR) on the SR Ca2+ content and contractions of single rat cadiomyocytes was investigated. In 80% of cells 10(-6) M TG blocked Ca2+ uptake by SR at rest, depleted the SR Ca2+, inhibited postrest potentiation and changed the pattern of response to premature contractions. Amplitude of steady state contractions was reduced to 11 +/- 4% of pre-TG control. In 20% of cells TG had similar effect on the SR function, however, amplitude of contractions was reduced to only 50 +/- 7% of control. It is concluded that the results obtained in 80% of the cells are compatible with the wide-spread notion that contractions of rat cardiac myocytes are activated predominantly by Ca2+ released from SR. However, population of myocytes of ventricular myocardium of this species is not homogeneous in this respect. In 20% of them contractions are activated by both sarcolemmal and SR Ca2+ contribution being approximately equal. These cells occupy intermediate position between guinea-pig cells and the bulk of rat cells.

The effect of menadione on sarcoplasmic reticulum Ca2+ and contractions of single guinea-pig cardiomyocytes.

We investigated the effect of 2-methyl-1,4-naphtoquinone (Menadione) on sarcoplasmic reticulum (SR) Ca2+ content and electrically stimulated contractions (ESCs) of single isolated myocytes of guinea-pig ventricular myocardium. The contractures initiated by means of microinjections of caffeine into the close vicinity of the cell were used as an indirect index of the SR Ca2+ content. Superfusion of the cells for 45 min with Menadione resulted in gradual disappearance of contractile responses to caffeine, prolongation of time to peak amplitude of ESCs by 48 +/- 15% and complete inhibition of postrest and postextrasystolic potentiation. These results are consistent with those of Floreani and Carpenedo (7) who found that Menadione strongly inhibits the SR Ca2+ ATPase. Despite depletion of the SR Ca2+ the amplitude of ESCs did not change which suggests that contractions were initiated in the cells treated with Menadione by Ca2+ derived from the sources other than the SR.

Calcium in the in situ mitochondria of rested and stimulated myocardium.

Calcium is believed to provide feedback between myocardial energy consumption and production. Calcium content was proved to increase in mitochondria (MT) isolated from (1) stimulated hearts, and (2) hearts of increased contractility. In this work we compared Ca2+ content in the intact MT of skinned strips excised at 0 degrees C from previously stimulated or rested guinea-pig ventricles equilibrated with 45Ca and in single rested or stimulated myocytes. In both preparations Ca2+ was released from MT by means of CCCP (carbonyl cyanide m-chlorophenyl-hydrazone; 100 microM). CCCP released 1.58 +/- 0.55 nmol Ca2+/mg of MT protein from the strips of rested hearts and 3.86 +/- 1.12 nmol Ca2+/mg of MT protein from the stimulated muscles. Stimulated myocytes responded to the close micro-injection of CCCP with transient contracture which was not inhibited by caffeine (10 mM) or ryanodine (0.1 microM, 45 min), although the time-course of the contracture was changed. Contracture could not be initiated in rested cells. It is suggested that in rested myocytes MT contain much less Ca2+ than in stimulated ones.

Method for isolation of adult mouse cardiac myocytes for studies of contraction and microfluorimetry.

We describe techniques for the isolation of Ca(2+)-tolerant myocytes from mouse (2 to 6 mo old) ventricle for measurements of mechanics of contraction and microfluorimetry. Our approach involved special modifications of existing methods that had been developed for other species but were not successful when applied to the mouse heart. Important features of the method are 1) a requirement for careful timing (< 5 min) of perfusion with nominally Ca(2+)-free solution; 2) perfusion with a solution containing a specially selected batch of collagenase in the presence of a low Ca2+ concentration; and 3) meticulous attention to water quality. Using this method, we could consistently isolate durable, Ca(2+)-tolerant myocytes from adult mouse hearts with a yield of approximately 50%. With slight modifications, the method should enable other investigators to isolate mouse cardiomyocytes for their specific experimental applications.

The effect of thapsigargin on sarcoplasmic reticulum Ca2+ content and contractions in single myocytes of guinea-pig heart.

Contractures initiated by 1 s superfusion of single myocytes of guinea-pig heart with 10 mM caffeine were used as a relative index of the SR Ca2+ content. Thapsigargin (Tg) in concentration 2 x 10(-7) M completely blocked Ca2+ uptake during electrical stimulation by the SR from which Ca2+ has been previously depleted by caffeine. Tg did not affect the SR Ca2+ content in the resting myocytes and did not block release of the SR Ca2+ during electrically stimulated contractions (ESCs). It is concluded that in guinea-pig myocytes Tg affects SR Ca2+ by selective blocking the SR Ca2+ uptake. The amplitude of steady state ESCs dropped to 68 +/- 5.4% (S.D., n = 20) of that of the pre-Tg control. Time to peak contraction increased from 454 +/- 82.4 ms to 820 +/- 157.4 ms and time of relaxation increased from 368 +/- 90.8 ms to 474 +/- 87 ms after the SR Ca2+ has been depleted by Tg. Rest decay of contractions, post-extrasystolic potentiation and post-rest potentiation were inhibited.

The role of sarcoplasmic reticulum and Na-Ca exchange in the Ca2+ extrusion from the resting myocytes of guinea-pig heart: comparison with rat.

Inhibition of the Na-Ca exchange at the beginning of rest in isolated myocytes of the guinea-pig heart by means of superfusion with Na,Ca-free solution or 5.0 mM Ni2+ resulted in appearance of multiple phasic contractures. Contractures could not be initiated when the sarcoplasmic reticulum (SR) Ca2+ had been depleted by short (1 s) or steady state exposure to 10 mM caffeine, 0.1 microM ryanodine or due to rapid spontaneous release of the SR Ca2+ occurring sometimes at the beginning of rest. Superfusion with 2 x 10(-7) M thapsigargin, which blocked the SR Ca2+ uptake, prevented contractures otherwise initiated by superfusion with the Na,Ca-free solution. The frequency of spontaneous contractures was positively related to the rate of stimulation before rest and negatively related to the duration of rest before superfusion with the Na,Ca-free solution. It is proposed that in guinea-pig myocardium Ca2+ taken up by the SR from sarcoplasm or other cellular compartments like mitochondria, is released during diastole and rest to the subsarcolemmal space from which it is extruded by means of Na-Ca exchange. The release is a primary event not dependent on decrease of the resting sarcoplasmic free [Ca2+] by the outward Ca2+ transport. Inhibition of the Na-Ca exchange at the beginning of rest did not initiate any contractile response in rat myocytes. If the spontaneous contractures were already present, they were inhibited by superfusion with the Na,Ca-free solution. The result reflects the basic difference in the properties of SR of guinea-pig and rat.

Net transsarcolemmal Ca2+ shifts versus Ca/Ca exchange in guinea pig ventricular muscle.

We investigated the net transsarcolemmal Ca2+ shifts and Ca/Ca exchange by means of 45Ca in isolated, perfused ventricles of guinea pig heart treated with vanadate to inhibit ATP-driven sarcolemmal Ca2+ pump. The heart was stimulated (at the rate of 60/min) and perfused with a solution containing 45Ca for 60 min. Thereafter stimulation was stopped and either perfusion with radioactive solution was continued or the solution was exchanged for a non-radioactive one. In the first case, tissue 45Ca content (equivalent to the exchangeable Ca2+ content) dropped from 1.960 +/- 0.120 mmol/kg of wet weight (w.w.) to 0.715 +/- 0.049 mmol/kg w.w. and stabilized at this level between 5th and 10th min. In the second case, decrease in 45Ca content continued and within 40 min attained 0.047 +/- 0.004 mmol/kg w.w., despite stabilizing of the total exchangeable Ca2+ content. Drop of 45Ca content in the rested heart perfused (until the end of experiments) with radioactive solution resulted from the net transsarcolemmal Ca2+ shift and it was strongly inhibited by removal of extracellular Na+. The continuing drop in 45Ca content in the heart perfused with non-radioactive solution while total Ca2+ content stabilized must have resulted from Ca/Ca exchange; it was stimulated by removal of extracellular Na+. These experiments separate two modes of 45Ca fluxes and suggest that a common route of these fluxes is the Na/Ca exchanger.

Quinolinic acid: effect on 45Ca content in perfused rat heart preparations and its calcium ion binding property in Krebs-Henseleit medium and blood serum.

In the experiment it was found that quinolinic acid perfusion was accompanied with the increase in calcium 45Ca content in myocardium tissue. The increase in calcium content was associated with a decrease in heart contractility. Moreover, it was shown that quinolinic acid can form complexes with calcium ions in Krebs-Henseleit medium as well as in blood serum but only to a small degree.

Quinolinic acid: a modulator of the heart calcium channel in the rat and a binder of calcium ions.

When rat heart preparations were perfused with quinolinic acid at a slow constant rate, a decrease in contractility was observed. A higher rate of perfusion resulted in a biphasic response, thus both a positive inotropic effect and then a decrease in heart contractility were visible. Using a polarographic method, the association constant of quinolinic acid with calcium ions (Ka) was found to be equal to 220. By combining the values from heart perfusion experiments with the calculated ones of free calcium ions, a linear correlation was obtained between the decreases of contractility and of calcium ions (r = 0.94).

The effects of blocking the Na-Ca exchange at intervals throughout the physiological contraction-relaxation cycle of single cardiac myocyte.

The method of rapid superfusion of the single isolated ventricular myocytes of guinea-pig heart was used in order to inhibit the Na-Ca exchange throughout the physiological contraction-relaxation cycle. Superfusion of the cell at selected intervals during the contraction with the Na,Ca-free solution resulted in increase in its amplitude, increase in time to peak shortening and in delay of relaxation, albeit the cells relaxed before reperfusion of normal Tyrode solution. The largest increase in amplitude of contraction (to 134 +/- 16%) was observed when the effective exchange of the cell's environment was attained approximately 50 ms after the pulse stimulating contraction. The effects declined promptly when the delay was increased beyond 100 ms. In the cells treated with 10 mM caffeine superfusion with the Na,Ca-free solution after the delay of 50-100 ms resulted in decrease in extent of shortening. Increase in delay resulted in slight increase in extent of shortening with respect to control and strong inhibition of relaxation. The strongest effects were observed when the delay was approximately 200 ms. Superfusion of the normal cells and of the cells treated with caffeine between contractions resulted in slight potentiation of the next beat. It is concluded that Na-Ca exchange provides an important mechanism of relaxation and outward Ca2+ transport in the physiological contraction of the ventricular cardiomyocyte.

Effect of ablation of phospholamban on dynamics of cardiac myocyte contraction and intracellular Ca2+.

We compared mechanical activity and Ca2+ transients of ventricular myocytes isolated from wild-type and phospholamban (PLB)-deficient mouse hearts in control conditions and during beta-adrenergic stimulation. Compared with wild-type controls, cells isolated from PLB-deficient mouse hearts showed 1) a 2-fold increase in extent of cell shortening, 2) a 3-fold increase in maximal shortening velocity, and 3) a 3.4-fold increase in maximal relengthening velocity. PLB-deficient myocytes also demonstrated significant increases in the peak amplitude of the fura 2 fluorescence ratio and the rates of rising and falling phases of the Ca2+ transient. The fura 2 diastolic ratios were similar in both groups, suggesting no change in intracellular Ca2+ during diastole. In PLB-deficient myocytes, 0.05 microM isoproterenol induced an increase in the twitch amplitude by 152 +/- 11% (n = 6) compared with 290 +/- 31% (n = 6) in wild-type cells. Maximal shortening velocity was increased by 183 +/- 10% (n = 6) in PLB-deficient myocytes, compared with 398 +/- 62% (n = 6) in wild-type cells. The isoproterenol-induced increase in maximum relengthening velocity was increased by 168 +/- 8% (n = 6) in PLB-deficient cells compared with 445 +/- 71% (n = 6) in wild-type myocytes. In both groups, these changes in contractile parameters were accompanied by changes in the Ca2+ transient. Our results indicate that phosphorylation of sites other than PLB may play an important role in regulation of contraction-relaxation dynamics of heart cells responding to beta-adrenergic stimulation.

Anti-adrenergic effects of nitric oxide donor SIN-1 in rat cardiac myocytes.

We studied how the nitric oxide (NO*) donor 3-morpholinosydnonimine (SIN-1) alters the response to beta-adrenergic stimulation in cardiac rat myocytes. We found that SIN-1 decreases the positive inotropic effect of isoproterenol (Iso) and decreases the extent of both cell shortening and Ca2+ transient. These effects of SIN-1 were associated with an increased intracellular concentration of cGMP, a decreased intracellular concentration of cAMP, and a reduction in the levels of phosphorylation of phospholamban (PLB) and troponin I (TnI). The guanylyl cyclase inhibitor 1H-8-bromo-1,2,4-oxadiazolo (3,4-d)benz(b)(1,4)oxazin-1-one (ODQ) was not able to prevent the SIN-1-induced reduction of phosphorylation levels of PLB and TnI. However, the effects of SIN-1 were abolished in the presence of superoxide dismutase (SOD) or SOD and catalase. These data suggest that, in the presence of Iso, NO-related congeners, rather than NO*, are responsible for SIN-1 effects. Our results provide new insights into the mechanism by which SIN-1 alters the positive inotropic effects of beta-adrenergic stimulation.

Changes in thyroid state affect pHi and Nai+ homeostasis in rat ventricular myocytes.

We have tested the hypothesis that thyroid state may influence both the flow of cellular Ca2+ and the myofilament response to Ca2+ by effects on intracellular pH (pHi) and Na+ (Nai+). Single cardiac myocytes isolated from hypothyroid, euthyroid and hyperthyroid animals were loaded with fura-2/AM (Cai2+ probe), BCECF/AM (pHi probe) or SBFI/AM (Nai+ probe). Compared with hypothyroid animals, myocytes isolated from hyperthyroid rat hearts demonstrated a significant: (1) increase in extent of shortening; (2) decrease in the time to peak contraction; (3) increase in the peak amplitude of the fura-2 fluorescence ratio; (4) decrease in pHi (DeltapHi=0. 19+/-0.05); and (5) increase in Nai+ (DeltaNai+=2.88+/-0.55 mM). We have also compared pHi in Langendorff perfused hypo- and hyperthyroid rat hearts using NMR. We have found that hyperthyroid hearts are 0.15+/-0.03 pH units more acidic than hypothyroid hearts. Analysis of mRNA levels demonstrated that hyperthyroidism increased expression of both the Na+/Ca2+ exchanger and Na+/H+ antiporter, and decreased expression of Na+ channel mRNAs. These changes appear partially responsible for the observed changes in Nai+ and pHi. Our results provide the first evidence that changes in cardiac contractility associated with altered thyroid state not only involve effects on Ca2+, but may also involve changes in the response of the myofilaments to Cai2+mediated by altered pHi and Nai+.

CGP-48506 increases contractility of ventricular myocytes and myofilaments by effects on actin-myosin reaction.

We measured the effects of the benzodiazocine derivative, CGP-48506 (5-methyl-6-phenyl-1,3,5,6-tetrahydro-3,6-methano-1, 5-benzodiazocine-2,4-dione), on contraction of intact myocytes and permeabilized fibers of rat ventricular muscle. CGP-48506 is unique in that it is able to sensitize cardiac myofilaments to Ca2+, but unlike all other agents in this class, it is not an inhibitor of type III phosphodiesterase. When added to isolated intact myocytes, CGP-48506 significantly increased the amplitude of cell shortening with little or no change in the Ca2+ transient, as determined by the fluorescence ratio of fura 2. The late phase of the relation between fura 2 ratio and cell length was shifted to the left in the presence of CGP-48506. CGP-48506 also induced a relatively small decrease in diastolic length. However, compared with the thiadiazinone EMD-57033, CGP-48506 had a much smaller effect on diastolic length at concentrations in which there was a bigger inotropic effect. When added to solutions bathing detergent-extracted (skinned) fiber bundles, CGP-48506 increased maximum force. CGP-48506 also increased submaximal force and shifted the pGa-force relation to the left. However, compared with EMD-57033, there was less of an effect of CGP-48506 on force at relatively high pCa values. CGP-48506 did not alter Ca2+ binding to myofilament troponin C. CGP-48506 was able to reverse inhibition of contraction induced by butanedione monoxime both in intact cells and in skinned fiber bundles. Our results indicate that CGP-48506, like EMD-57033, is a positive inotropic agent working through a direct effect downstream from troponin C. CGP-48506, however, appears to have a unique mechanism resulting in less effect on diastolic function.

Increased contractility and altered Ca(2+) transients of mouse heart myocytes conditionally expressing PKCbeta.

Activation of protein kinase C (PKC) in heart muscle signals hypertrophy and may also directly affect contractile function. We tested this idea using a transgenic (TG) mouse model in which conditionally expressed PKCbeta was turned on at 10 wk of age and remained on for either 6 or 10 mo. Compared with controls, TG cardiac myocytes demonstrated an increase in the peak amplitude of the Ca(2+) transient, an increase in the extent and rate of shortening, and an increase in the rate of relengthening at both 6 and 10 mo of age. Phospholamban phosphorylation and Ca(2+)-uptake rates of sarcoplasmic reticulum vesicles were the same in TG and control heart preparations. At 10 mo, TG skinned fiber bundles demonstrated the same sensitivity to Ca(2+) as controls, but maximum tension was depressed and there was increased myofilament protein phosphorylation. Our results differ from studies in which PKCbeta was constitutively overexpressed in the heart and in studies that reported a depression of myocyte contraction with no change in the Ca(2+) transient.

Altered hemodynamics in transgenic mice harboring mutant tropomyosin linked to hypertrophic cardiomyopathy.

We used transgenic (TG) mice overexpressing mutant alpha-tropomyosin [alpha-Tm(Asp175Asn)], linked to familial hypertrophic cardiomyopathy (FHC), to test the hypothesis that this mutation impairs cardiac function by altering the sensitivity of myofilaments to Ca(2+). Left ventricular (LV) pressure was measured in anesthetized nontransgenic (NTG) and TG mice. In control conditions, LV relaxation was 6,970 +/- 297 mmHg/s in NTG and 5,624 +/- 392 mmHg/s in TG mice (P < 0.05). During beta-adrenergic stimulation, the rate of relaxation increased to 8,411 +/- 323 mmHg/s in NTG and to 6,080 +/- 413 mmHg/s in TG mice (P < 0.05). We measured the pCa-force relationship (pCa = -log [Ca(2+)]) in skinned fiber bundles from LV papillary muscles of NTG and TG hearts. In control conditions, the Ca(2+) concentration producing 50% maximal force (pCa(50)) was 5.77 +/- 0.02 in NTG and 5.84 +/- 0.01 in TG myofilament bundles (P < 0.05). After protein kinase A-dependent phosphorylation, the pCa(50) was 5.71 +/- 0.01 in NTG and 5.77 +/- 0. 02 in TG myofilament bundles (P < 0.05). Our results indicate that mutant alpha-Tm(Asp175Asn) increases myofilament Ca(2+)-sensitivity, which results in decreased relaxation rate and blunted response to beta-adrenergic stimulation.