Rapid atrial pacing produces atrial systolic and diastolic failure characterized by absent atrial booster pump function, increased atrial chamber stiffness, enhanced atrial conduit function, and atrial enlargement. However, the processes underlying these abnormalities are poorly understood. Therefore, we examined left atrial myocardium from dogs with rapid pacing-induced atrial failure (400 bpm for 6 weeks) and from control dogs. Western blotting was used to determine the levels of proteins involved in calcium homeostasis (SERCA 2A, phospholamban, Na + -Ca 2+ exchanger). Matrix metalloproteinase (MMP) activity was measured using gelatin and casein zymography, and levels of tissue inhibitor of metalloproteinase-4 (TIMP-4) and the TIMP-4 complexed with MMPs were measured with Western blot analysis. There were no differences in SERCA 2A or Na + -Ca 2+ exchanger protein levels, but phospholamban level was significantly decreased in atrial samples from rapidly paced dogs (51.2 ± 7.8 vs. 77.0 ± 10.0, p < 0.01). The activity of MMP-9 was selectively and significantly increased by ∼ 50%, and the level of complexed TIMP-4 protein was significantly decreased by ∼ 50% in samples from dogs with atrial failure. Thus, rapid pacing-induced atrial failure is associated with differential changes in MMP activity, an unchanged number of calcium pumps, and compensatory changes in the level of phospholamban.

The objective of this study was to characterize genetic variation in complex cardiovascular traits in two commonly used inbred mouse strains. We performed echocardiography, graded treadmill exercise, tail cuff plethysmography, and telemetry (heart rate, activity, temperature) in age- (∼9 weeks) and sex-matched A/J and C57BL/6J (B6) inbred mice. B6 mice had significantly larger end-diastolic dimension (3.31±0.42 mm versus 2.83±0.31 mm) and left ventricle mass (46.2±14.1 versus 32.7±11.5 g) than A/J mice. This relative hypertrophy was eccentric (relative wall thickness ratios: 0.30±0.01 versus 0.32±0.01) and was not associated with a difference in systolic blood pressure (122.0±13.2 versus 123.1±20.8 mmHg). Left ventricle fractional shortening (39.1±6.2 versus 47.1±6.9%) and heart rate (433±55 versus 524±45 beats per minute) were significantly lower in B6 versus A/J, respectively, resulting in similar resting echocardiographic cardiac indices (0.58±0.19 versus 0.50±0.17 ml/min/g). Maximum exercise time on a treadmill was significantly greater in B6 than in A/J mice (9.6±3.4 versus 4.4±1.9 minutes). Telemetry showed that body temperature was generally greater and heart rate lower in B6 than A/J; the relation with activity was more complex. These data suggest that relative to A/J, B6 mice have a phenotype characteristic of the “athlete's heart,” that is, eccentric, physiologic hypertrophy, slower heart rates, and increased exercise endurance. This systematic characterization of functionally related cardiovascular traits in A/J and C57BL/6J mice revealed numerous differences whose genetic bases can be dissected with recombinant inbred, recombinant congenic, and chromosome substitution strains.

Objective: M‐mode echocardiography has been used extensively for estimating left ventricular (LV) mass in mice, but accuracy is limited by its unidimensional nature. This study tested whether two‐dimensional (2‐D) echocardiographic measurement of LV mass using area‐length and truncated ellipsoid methods overcomes this limitation. Methods: Twenty‐five anesthetized FVB/N mice (Mus musculus) underwent high resolution (15 MHZ) two‐dimensionally directed M‐mode and 2‐D echocardiography. End‐diastolic epicardial and endocardial areas from short‐axis views and the LV orthogonal long‐axis views were measured, while LV mass was calculated by 2‐D area‐length and truncated ellipsoid methods. LV mass was calculated from M‐mode using the uncorrected cube equation. Hearts were removed and weighed after completing the echocardiographic studies. The correlation and agreement between echocardiographic‐determined LV mass and gravimetric LV weight were examined. Results: LV mass using the area‐length method correlated better with autopsy LV weight (r = 0.87, slope = 0.87, standard error of the estimate [SEE]= 12.4 mg, P < 0.001) than M‐mode echocardiography (r = 0.85, slope = 0.71, SEE = 11.1 mg, P < 0.001), and the degree of agreement (bias ± 2 SD) was better (10.2 ± 25.1 mg vs 13.1 ± 25.8 mg, P NS). Although 2‐D truncated ellipsoid LV mass showed a good correlation with necropsy LV weight (r = 0.87, slope = 0.78, SEE = 11.0 mg, P < 0.001), the agreement between the truncated ellipsoid estimates and LV weight was less than that between M‐mode and gravimetric LV mass. Conclusions: The 2‐D area‐length method is an accurate method of LV mass determination in symmetrical ventricles and can be used as an alternative for in vivo measurement of LV mass in mice.

Iron-overload cardiomyopathy is the most common cause of death in patients with thalassemia major, yet the associated changes in cardiac function have not been quantified. We studied the effects of iron overload on cardiac function in Mongolian gerbils, a species that responds to iron overload in the same manner as human beings. We injected iron-dextran or dextran alone at low subcutaneous doses (200 mg/kg/wk) for 20 to 60 weeks and at high doses (800 mg/kg/wk) for 6 to 20 weeks. At shorter durations for either dose, the mean values of cardiac work, coronary flow, left ventricular (dP/dt)(max) and left ventricular (dP/dt)(min) in isolated perfused hearts were significantly greater than control values; at longer durations, these values were significantly less than control values. Echocardiography in intact animals showed eccentric cardiac hypertrophy, increased cardiac output, and normal exercise tolerance at shorter durations of dosage. At longer durations, concentric cardiac hypertrophy developed, and cardiac output and exercise capacity were impaired. The response to iron overload in Mongolian gerbils progresses from an initial state of high cardiac output to a subsequent state of low-output failure similar to the course of cardiomyopathy that has been inferred in patients with transfusional iron overload.

Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

Ectopic expression of the sarcoplasmic reticulum (SR) Ca 21 ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca 21 transients; however, the cellular mechanisms underlying altered Ca 21 handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca 21 load measurements revealed that TG myocytes have significantly enhanced SR Ca 21 lo d. Confocal line-scan images of field-stimulated SR Ca release showed an increased rate of Ca 21 removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by '30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca 21 current density was reduced by '50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca 21 transport and cardiac contractility. (Circ Res. 2001;89:160-167.)

OBJECTIVE:Left ventricular assist device support mechanically unloads the failing ventricle with resultant improvement in cardiac geometry and function in patients with end-stage heart failure. Activation of the G alpha q signaling pathway, including protein kinase C, appears to be involved in the progression of heart failure. Similarly down-regulation of Ca2+ cycling proteins may contribute to contractile depression in this clinical syndrome. Thus we examined whether protein kinase C activation and decreased Ca2+ cycling protein levels could be reversed by left ventricular assist device support. METHODS:Left ventricular myocardial specimens were obtained from seven patients during placement of left ventricular assist device and heart transplantation. We examined changes in protein levels of G alpha q, phospholipase C beta 1, regulators of G protein signaling (RGS), sarcoplasmic reticulum Ca2+ ATPase, phospholamban and translocation of protein kinase C isoforms (alpha, beta 1, and beta 2). RESULTS:The paired pre- and post-left ventricular assist device samples revealed that RGS2, a selective inhibitor of G alpha q, was decreased (P < 0.01), while the status of G alpha q, phospholipase C beta 1, RGS3 and RGS4 were unchanged after left ventricular assist device implantation. Translocation of protein kinase C isoforms remained unchanged. Left ventricular assist device support increased sarcoplasmic reticulum Ca2+ ATPase protein level (P < 0.01), while phospholamban abundance was unchanged. CONCLUSIONS:We conclude that altered protein expression and stoichiometry of the major cardiomyocyte Ca2+ cycling proteins rather than reduced phospholipase C beta 1 activation may contribute to improved mechanical function produced by left ventricular assist device support in human heart failure.

Dilated cardiomyopathy is characterized by decreased contractile function and loss of myofibril organization. Previously unexplored structural and molecular events that precede and initiate dilation can now be studied in tropomodulin-overexpressing transgenic (TOT) mice exhibiting progressive dilated cardiomyopathy, Onset of dilation did not correspond to a change in transgene expression levels, which were more than threefold above normal at birth and remained elevated throughout postnatal life. Similarly, mitogen-activated protein kinase activation (p38, ERK1/ERK2, JNK1/JNK2) was not associated with dilation. In contrast, calcineurin was activated before dilation, presumably due to doubling of intracellular diastolic calcium levels in TOT cardiomyocytes, Amplitude of systolic calcium transients was greatly increased as well, demonstrating the novel and unique calcium handling profile of TOT cardiomyocytes, Loss of myofibril organization was not apparent by confocal microscopy until over 1 week after birth, although neonatal sarcomeric abnormalities were revealed by ultrastructural analysis. Rapid postnatal increases in heart:body weight ratio at 1.5 weeks were followed by two waves of mortality between 2 and 3 weeks after birth coincident with maturational stress. Ultimately, TOT pathogenesis is a compensatory response to altered sarcomeric structure driven by calcineurin activation within days after birth, making TOTs an excellent paradigm for studying the role of calcium overload in dilated cardiomyopathy.

Annexins are a unique family of calcium-dependent, phospholipid-binding proteins found in various tissues. Annexin VI, a major member in the family, exists in the heart and acts as a potent regulator of the sarcoplasmic reticulum calcium-release channel, cardiac L-type calcium channel and Na+/Ca2+ exchanger. To investigate the role of the regUlation of annexin VI in intracellular calcium homeostasis and mechanical properties of cardiomyocytes. we used the transgenic mice which overexpresses annexin VI (TG, n = 6) and control mice (C, n = 6) at three months of age. A 10-fold increase in annexin VI was revealed by Western blotting in TG mice compared to the controls. No differences were found between TG and C groups in heart/body wt., lung/body wt. or myocyte size. Cardiomyocytes from TG and C mice were isolated and field paced at 0.25, 0.5 and 1.0 Hz (15,30 and 60 beats per minute). The myocytes of TG mice demonstrated significant decreases in shortening fraction (% shortening, TG vs C, 4.59 ± 0.42 vs 8.62 ± 0.85, P < 0.01), rate of shortening (+dl/dt,μm/sec, TG vs C, 93.54 ± 24.31 vs 148.77 ± 20.23, P < 0.05) and rate of relengthening (-dl/dt.μm/sec, TG vs C, 59.28 ± B.32 vs 119.66 ± 15.94. P < 0.01) at 1.0, but not at 0.25 or 0.5 Hz stimulation. FURA-2 loaded myocytes from TG mice showed lower baseline levels of calcium at all three stimulation frequencies respectively (340/380 ratio, TG vs C, 0.526 ± 0.075 vs 0.968 ± 0.550 at 0.25 Hz, P < 0.001; 0.566 ± 0.044 vs 0.9B2 ± 0.050 at 0.5 Hz, P < 0.001; 0.564 ± 0.043 vs 1.016 ± 0.051 at 1.0 Hz, P < 0.001). The amplitude of calcium signals decreased in annexin VI TG mice at 0.25 Hz (340/380 ratio. TG vs C, 0.974 ± 0.14B vs 0.541 ± 0.082, P < 0.05) and at 0.5 Hz stimulation (0.858 ± 0.141 vs 0.503 ± 0.077, P < 0.05). The duration of calcium signals was attenuated in TG mice compared to control at 0.25 Hz (sec, 1.033 ± 0.227 vs 1.507 ± 0.150, P < 0.05) We conclude that overexpression of annexin VI in mouse myocytes causes depression in the intracellular free calcium concentration. and frequencydependent depression in the extent and rate of the shortening and relengthening.