Accelerated Cardiomyopathy in Mice With Overexpression of Cardiac G s and a Missense Mutation in the -Myosin Heavy Chain

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1 Accelerated Cardiomyopathy in Mice With Overexpression of Cardiac G s and a Missense Mutation in the -Myosin Heavy Chain Stefan E. Hardt, MD; Yong-Jian Geng, MD, PhD; Olivier Montagne, MD; Kuniya Asai, MD, PhD; Chull Hong, MD; Gui Ping Yang, MD; Sanford P. Bishop, DVM, PhD; Song-Jung Kim, PhD; Dorothy E. Vatner, MD; Christine E. Seidman, MD; J.G. Seidman, PhD; Charles J. Homcy, MD; Stephen F. Vatner, MD Background To understand further the pathogenesis of familial hypertrophic cardiomyopathy, we determined how the cardiomyopathy induced by an Arg4033Gln missense mutation in the -myosin heavy chain (403) is affected by chronically enhancing sympathetic drive by mating the mice with those overexpressing G s (G s x403). Methods and Results Heart rate in 3-month-old conscious mice was elevated similarly (P 0.05) in mice overexpressing G s (G s mice; bpm) and G s x403 mice ( bpm) compared with littermate wild-type mice (WT; bpm) and 403 mice ( bpm). Left ventricular ejection fraction (LVEF), as determined by echocardiography, was enhanced in G s x403 mice (88 1%, P 0.001) compared with WT (69 1%), 403 (75 1%), and G s (69 2%) mice. Isolated cardiomyocytes from G s x403 mice also exhibited higher (P 0.001) baseline percent contraction ( %) than WT ( %), 403 ( %), and G s ( %) cardiomyocytes. Relaxation of myocytes was impaired in 403 mice compared with WT but enhanced in G s and normalized in G s x403 mice. This was also observed in vivo. In vivo isoproterenol (0.1 g kg 1 min 1 ) increased LVEF to maximal levels in G s x403 and G s, whereas in 403, the response was attenuated compared with WT. At 10 months of age, G s x403 had significantly depressed LVEF (57 4%). Histopathological examination demonstrated that myocyte hypertrophy and fibrosis were already present in young G s x403 mice and that old animals had severe cardiomyopathy. By 15 months of age, the survival of G s x403 was 0% compared with 100% for WT, 71% for G s, and 100% for 403 mice (P 0.05). Conclusions These results show that the cardiomyopathy developed by G s x403 mice is synergistic rather than additive, most likely owing to the elevated baseline function combined with enhanced responsiveness to sympathetic stimulation. (Circulation. 2002;105: ) Key Words: hypertrophy cardiomyopathy heart failure Familial hypertrophic cardiomyopathy (FHC) is considered to be a disease of the sarcomere, 1,2 and several mutations in different sarcomeric proteins have been identified in affected individuals. 3 5 In some cases, FHC is associated with premature death and frequent arrhythmias, 6 but why this occurs in some patients and not in others is not known. Indeed, even though the genetic mutations have been widely identified, the pathophysiological mechanisms leading to the clinical phenotype are still poorly understood. Genetically altered mice bearing myosin heavy chain mutations that mimic FHC in humans are a useful model to clarify the processes that lead to the common phenotype of FHC, eg, mice with an Arg4033 Gln missense mutation in the -myosin heavy chain (403 mice), 7 which appear morphologically normal when they are young but develop hypertrophy and cardiomyopathy when they age. 8 On the other hand, transgenic mice with overexpression of G s (G s mice) exhibit a hyperadrenergic phenotype (specifically, enhanced contractile and relaxation responses to -adrenergic stimulation) but remarkably also develop cardiomyopathy when they age. 9,10 The goal of this investigation was to determine whether a new mouse model created by mating 403 mice with G s mice (G s x403) affected the development of cardiomyopathy in these mice and specifically (1) whether the cardiomyopathy was accelerated, (2) whether the diastolic dysfunction in 403 Received September 10, 2001; revision received October 30, 2001; accepted November 19, From the Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine and Department of Medicine, University of Medicine and Dentistry New Jersey, New Jersey Medical School, Newark (S.E.H., O.M., K.A., C.H., G.P.Y., S.-J.K., D.E.V., S.F.V.); University of Texas-Houston Medical School (Y.-J.G.); University of Alabama at Birmingham (S.P.B.); Harvard Medical School, Boston, Mass (C.E.S.); Howard Hughes Medical Institute, Boston, Mass (J.G.S.); and COR Therapeutics, South San Francisco, Calif (C.J.H.). Guest Editor for this article was Robert Roberts, MD, FACC, FRSM, FRC, Baylor College of Medicine, Houston, Tex. Correspondence to Stephen F. Vatner, MD, Cardiovascular Research Institute, New Jersey Medical School, University of Medicine and Dentistry New Jersey, 185 S Orange Ave, Medical Science Bldg I-576, Newark, NJ vatnersf@umdnj.edu 2002 American Heart Association, Inc. Circulation is available at 614

2 Hardt et al Accelerated Cardiomyopathy in G s x403 Mice 615 mice was corrected, and (3) whether -adrenergic responsiveness was altered in dual transgenic animals, in view of the fact that -adrenergic responsiveness is enhanced in G s 10 and depressed in 403 mice (unpublished observations). To address these goals, we examined G s x403 mice in vivo using echocardiography and left ventricular (LV) pressure measurements to assess LV function and in vitro using isolated cardiomyocytes, and we performed postmortem histological examinations of the hearts. These results could potentially explain why some individuals with FHC fare more poorly than others. Methods Transgenic Animals G s -transgenic mice, which overexpress cardiac G s 3- to 5-fold, were mated with heterozygous 403 mice to produce a dual transgenic mouse line. Four groups of the first-generation offspring were generated, including wild-type (WT) mice, G s -overexpressing mice, 403 mice, and G s x403 mice, with each accounting for 25% of the total offspring, as confirmed by genotyping by methods described previously. 7,9 Thus, the WT littermates were true controls for the G s x403 mice. Initial studies were performed in 3- to 4-monthold mice. To determine changes in LV function over time, a total of 32 mice from the 4 groups (6 to 9 per group) were examined by echocardiography at 3 and 6 months of age; thereafter, the same mice were studied serially each month by echocardiography up to 10 months of age. Survival was followed up in these mice up to 15 months of age. Histological analysis was performed in 3- to 4-month-old and in 10- to 11-month-old mice. Heart Rate in Conscious Mice Heart rate was measured by telemetry techniques under conscious and unrestrained conditions in 3-month-old animals. Mice were anesthetized with an intraperitoneal injection of ketamine (0.065 mg/g), acepromazine (0.02 mg/g), and xylazine (0.013 mg/g), and a telemetry transducer (TA10EA-F20; Data Science Co) was implanted subcutaneously into the back, with paired electrodes placed around the thorax. Experiments were initiated 3 to 5 days after recovery from surgical instrumentation. Mice with implanted telemetry devices were housed in individual cages with free access to food and water and were exposed to 12-hour-light/12-hour-dark cycles. Echocardiography Mice were anesthetized as described above. The procedure for echocardiography has been reported previously To assess responses to -adrenergic stimulation, LV function was measured during intravenous infusion of isoproterenol (0.1 g kg 1 min 1 for 5 minutes) via a chronically implanted jugular catheter. LV Hemodynamics and Arterial Pressure The mice (3 months old) were anesthetized as described above, and a bipolar transesophageal pacing electrode was introduced. A 1.4F micromanometer catheter (Millar Instruments Inc) was inserted into the right carotid artery, and LV hemodynamics and arterial blood pressures were measured at spontaneous heart rates and during atrial pacing with 550 bpm. Measurement of Contractile and Relaxation Function Cardiac myocytes were prepared as described previously. 13,14 Myocytes were transferred to a warmed and continuously perfused cell chamber located on an inverted microscope stage (Nikon Inc). Myocyte length was measured with a video motion edge detector (VED103; Crescent Electronics), and the data were acquired at 240 images per second. Myocyte contraction was also examined in response to isoproterenol (10 8 mol/l) to determine the extent to which -adrenergic function is altered in G s x403 mice. To examine whether the inotropic response of myocytes to non -adrenergicreceptor mediated stimulation was altered, myocyte function was assessed with Ca 2 (4 mmol/l). Myocyte contractile and relaxation function was also assessed with Rp-cAMP (200 mol/l), which blocks protein kinase A. Myocytes were loaded with 3.8 mmol/l of fura-2 (Sigma Chemical Co), and intracellular free Ca 2 was measured as the fluorescence ratio (340/380 nm). 13,14 Pathology Histological studies of the hearts were conducted in perfusion-fixed hearts from 28 younger mice (3 to 4 months old) and 24 older mice (10 to 11 months old). Myocardial connective tissue was analyzed quantitatively in a cross section of the LV obtained midway between base and apex and stained with picric acid sirius red F36 as described previously. 12 DNA fragmentation of myocyte nuclei was detected in situ by use of terminal dutp nick end-labeling (TUNEL) on paraffin sections of the mouse hearts, as described previously. 15 Biochemistry Western blot analysis of G s and -receptor density was performed from mouse hearts as described previously. 16 Statistical Analysis All data were reported as mean SEM. Group comparisons were analyzed by multiple analysis of variance (MANOVA), followed by post hoc comparisons between groups with the least significant difference test or by ANOVA for repeated measurements. A P value 0.05 was considered statistically significant. Results In Vivo Physiology Heart rate (bpm) measured in the conscious state was elevated similarly (P 0.05) in 3-month-old G s (746 14) and G s x403 (718 19) mice compared with either WT (623 18) or 403 (594 16, n 6 to 10 per group) mice. Heart rate was also elevated in these 2 groups compared with WT and 403 mice under anesthesia (Table 1). LV dp/dt max (mm Hg/s), measured in anesthetized animals, was enhanced only in G s x403 mice ( , P 0.05; Table 2) compared with WT ( ), G s ( ), and 403 ( ) mice. To exclude potential confounding effects of different heart rates, measurements were repeated during transesophageal pacing at 550 bpm. With heart rate held constant, LV dp/dt max (mm Hg/s) was still only higher (P 0.05) in G s x403 mice ( ) compared with WT ( ), G s ( ), and 403 ( ) mice. Impaired relaxation (LV dp/dt min, mm Hg/s) in anesthetized 403 mice in spontaneous rhythms ( ) was restored in G s x403 ( , P 0.05; Table 1). Ejection fraction was one LV functional parameter used, recognizing that there are limitations to its interpretation because only 1 LV axis was measured. In G s x403 mice, LV ejection fraction (LVEF) was enhanced in young animals and depressed by 10 months of age (Figure 1). LVEF was enhanced in G s x403 mice (88 1%, P 0.001) compared with WT (69 1%), 403 (75 1%), and G s (69 2%) mice (Table 1). The Vcfc (velocity of circumferential fiber shortening corrected for heart rate) versus afterload relation also revealed enhanced contractility in G s x403 mice compared with all other groups (Figure 2, left). Relaxation half-time, the time required for LV pressure at end ejection to be reduced by 50%, which is another index of diastolic function, was prolonged in 403 mice and shortened in G s and G s x403

3 616 Circulation February 5, 2002 TABLE 1. Baseline Hemodynamics and Echocardiography in Anesthetized 3-Month-Old Mice WT 403 G s G s x403 Hemodynamics Heart rate, bpm * * * Ao mean,mmhg LV dp/dt max, mm Hg/s LV dp/dt min, mm Hg/s * * * LVP sys,mmhg LVP dias,mmhg Number of animals Echocardiography Heart rate, bpm * * Ejection fraction, % * Fractional shortening, % * * EDD, mm ESD, mm * Number of animals Ao mean indicates mean systolic aortic pressure; LVP sys, systolic left ventricular pressure; LVP dias, end-diastolic left ventricular pressure; EDD, end-diastolic left ventricular diameter; and ESD, end-systolic left ventricular diameter. *P 0.05 vs WT; P 0.05 vs all other groups. mice (Figure 2, right). At 6 months of age, echocardiographic measurements were unchanged, whereas at 10 months of age, LVEF was depressed significantly only in G s x403 mice (57 4%) compared with LVEF in these same mice at 6 months of age (Figure 3). LV end-diastolic diameter was similar among groups at 3 months of age (Table 1). By 10 months of age, only the G s x403 mice exhibited LV dilatation as reflected by LV end-diastolic diameter, which was significantly higher ( mm) than for 403 ( mm), G s ( mm), and WT ( mm) mice (P 0.05), but end-diastolic LV anterior wall thickness TABLE 2. was significantly increased only in 403 mice. Isoproterenol (0.1 g kg 1 min 1 ) increased LVEF to maximal levels in G s x403 (91 1%) and G s (90 2%) mice but not in 403 (80 1%) or WT (80 2%) mice (Figure 4, left). Mortality By 15 months of age, mortality was 100% in G s x403 mice compared with 0% in WT, 29% in G s, and 0% in 403 mice (P 0.05). Postmortem analysis revealed overt signs of heart failure (ascites, pleural effusion, and enlargement of LV and right ventricle) in all G s x403 mice that died spontaneously. Morphology, Function, and Ca 2 Handling in Isolated Cardiomyocytes WT 403 G s G s x403 Diastolic length, m Systolic length, m Contraction, % TR70, ms * 62 3 dl/dt max, m/s * * Number of animals Number of myocytes Ca 2 transients Baseline Maximum value Difference * TR70, ms * * 111 4* Number of animals Number of myocytes TR70 indicates time to 70% of end-diastolic value. *P 0.05 vs WT; P 0.05 vs all other groups. Statistics based on number of animals.

4 Hardt et al Accelerated Cardiomyopathy in G s x403 Mice 617 Figure 1. LV function deteriorates in G s x403 mice over course of time. A, Example of M-mode tracing in 3-monthold WT mouse. B, M-mode tracing from same G s x403 mouse at 3 months of age displaying enhanced function (top) and at 10 months of age showing enlargement of LV and depressed LV function. Isolated Cardiomyocytes In parallel to the in vivo findings, G s x403 cardiomyocytes had a significantly higher baseline contraction ( %) than WT ( %), 403 ( %), and G s ( %) cardiomyocytes (Table 2). Consistent with the in vivo findings, the time of relaxation required for myocytes to lengthen to 70% of end-diastolic baseline, an indicator of lusitropic properties, was prolonged (P 0.05) in 403 (82 6 ms) compared with WT (68 4 ms) mice but normalized in G s x403 mice (62 3 ms) and enhanced (P 0.05) in G s mice (56 3 ms; Table 2). The maximal rate of relengthening ( dl/dt max ), another index of diastolic properties, was enhanced in G s and G s x403 mice but diminished in 403 mice (Table 2). Similar to in vivo results, isoproterenol (10 8 mol/l) also elicited enhanced responsiveness in isolated myocytes from G s and G s x403 mice and a blunted response to -adrenergic stimulation in 403 myocytes (Figure 4, middle). In contrast, nonspecific inotropic stimulation with increased extracellular Ca 2 (4 mmol/l) increased percent contraction similarly in all groups (Figure 4, right). Baseline contractility after incubation with Rp-cAMP did not significantly change in G s x403 myocytes ( %) and was still higher than percent contraction of WT ( %), G s ( %), and 403 ( %) myocytes. Ca 2 transients were significantly higher in G s x403 mice than in WT and 403 mice (Table 2). There was no difference among groups for diastolic Ca 2 content of the myocytes. Ca 2 reuptake, as assessed by the time for recovery to 70% of baseline, was prolonged in 403 myocytes ( ms) compared with WT myocytes (138 7 ms) and was shorter in G s ( ms) and G s x403 (111 4 ms) myocytes. Biochemistry G s -protein was upregulated in G s ( ) and G s x403 ( ) mice compared with WT mice ( ) and was consistently downregulated in 403 mice ( ; P 0.05, n 10 animals per group). In contrast, G i protein remained unchanged among groups (data not shown). -Receptor density was unchanged in 403 mice (WT, fmol/mg; 403, fmol/mg). Figure 2. Left, Vcfc vs afterload as an index of LV contractility is enhanced only in G s x403 mice. n 4 to 6 animals per group. Right, Relaxation half-time is prolonged in 403 mice compared with WT but reduced in G s and G s x403 mice. n 11 to 13 animals per group. *P 0.05 vs WT.

5 618 Circulation February 5, 2002 Figure 3. Ejection fraction as determined by echocardiography over course of time. Recordings are derived from serial measurements in same animals. G s x403 mice have enhanced LV function at 6 months of age compared with all other groups. At 10 months of age, cardiomyopathy developed, and LV function was depressed compared with WT. *P 0.05 vs 6 months. n 6 to 9 animals per group. Data at 3 months of age are shown in Table 1. Pathology Heart weight/body weight ratio (mg/g) of fresh tissue was significantly higher (P 0.05) in 3-month-old G s x403 mice ( ) than in WT ( ), G s ( ), and 403 ( ) mice, whereas body weight was similar among the groups. Histopathological examination demonstrated that mild to moderate myocyte hypertrophy and fibrosis were present in 3-month-old G s x403 mice, whereas lesions were essentially absent in the other groups (data not shown). Heart weight/body weight ratio was significantly increased in 10- to 11-month-old G s x403 mice ( ) compared with WT ( ), G s ( ), and 403 ( ) mice. Among the 10- to 11-month-old mice (Figure 5), the WT mice appeared normal and the 403 group had occasional focal lesions of increased interstitial fibrosis, enlarged myocytes, and evidence of myofiber disarray in ventricular myocytes, similar to that reported previously. 7 The G s group also had lesions similar to those reported previously in mice aged 10 months or older, 12 including multifocal regions of interstitial fibrosis; myocyte enlargement; enlarged bizarre nuclei, sometimes with vacuoles; focal cytoplasmic vacuoles in myocytes; and evidence of myocyte degeneration, atrophy, and cell loss. These lesions were markedly accentuated in the G s x403 mice (Figure 5d). Myocyte size was increased in G s and 403 mice compared with WT and was greater in G s x403 mice than in all other groups (Figure 6A). Quantitative evaluation Figure 5. Myocyte hypertrophy and fibrosis in 10- to 11-monthold animals. Photomicrographs of LV myocardium from 10- to 11-month-old WT (a), 403 (b), G s (c), and G s x403 (d) mice stained with hematoxylin and eosin. In both G s and G s x403 mice, there was myocyte hypertrophy, increased interstitial fibrosis, and cellular degeneration. Note enlarged bizarre nuclei (arrows), cytoplasmic vacuoles (arrowheads), and pale degenerating myocytes (double arrows). Scale bar in c is 50 m and same for all. of volume percent collagen (Figure 6B) revealed a large increase in the G s x403 mice compared with the other groups. The percent of TUNEL-positive myocytes was also markedly increased in G s x403 mice (Figure 6C, indicating enhanced apoptosis). Discussion We investigated the effects of targeted overexpression of G s in the hearts of 403 mice, a transgenic animal model of FHC. Our results indicate that the mechanisms that cause cardiomyopathy in G s and 403 mice act in concert in the development of cardiomyopathy, which is accelerated and more severe in G s x403 mice than in either of the parents and ultimately leads to premature death in these animals, potentially offering an explanation for the adverse consequences in some patients with FHC but not others. The G s x403 mice exhibit evidence of beginning cardiomyopathy, eg, fibrosis and hypertrophy, at 3 months of age, which becomes more severe and includes depressed LV function, LV dilatation, and increased apoptosis as the animals age. There are several possible explanations for the mechanism of this accelerated Figure 4. Left, Change in ejection fraction during intravenous isoproterenol infusion (Iso) from baseline (BL). -Adrenergic response to Iso is depressed in 403 mice compared with WT and enhanced in G s mice compared with WT. n 4 to 8 animals per group. Middle, In isolated myocytes, increases in percent contraction to Iso are blunted in 403 mice and enhanced in G s and G s x403 mice. n 5 to 7 animals per group. Right, Increases in percent contraction to stimulation with Ca 2 4 mmol/l were not different among groups. n 3 to 5 animals per group. *P 0.05 vs WT.

6 Hardt et al Accelerated Cardiomyopathy in G s x403 Mice 619 Figure 6. Quantitative histopathological analysis of hearts from 10- to 11-month-old animals. A, Quantification of myocyte cross-sectional area. Cross-sectional area was increased in both G s and G s x403 mice compared with WT. n 5 to6animals per group. B, Quantitative analysis of percent collagen in different groups. Collagen was highest in G s x403 mice but was also increased in G s mice. n 6 animals per group. C, Apoptosis as determined by TUNEL staining was higher in G s and G s x403 mice than in WT. n 6 animals per group. *P 0.05 vs WT, #P 0.05 vs all other groups. cardiomyopathy. The most intriguing possibility relates to the enhanced cardiac function in vivo and myocyte function in vitro at baseline and in response to -adrenergic stimulation. Impaired relaxation and blunted responses to -adrenergic stimulation characteristic of 403 mice were not only corrected but actually enhanced in the G s x403 mice in vivo. Similar findings were observed in isolated cardiomyocytes, ie, enhanced contraction and relaxation in G s x403 myocytes, which was also reflected in the Ca 2 transients. These findings in isolated myocytes are important because this occurs in the absence of neurohormonal stimulation. The increased baseline levels of heart rate, systolic function, and diastolic function, in combination with enhanced -adrenergic responsiveness, provide a framework for understanding the mechanism of the earlier and more severe development of cardiomyopathy. The question arises, why is baseline function enhanced in G s x403 compared with G s alone? The answer to this question will be essential for understanding the mechanism of the accelerated cardiomyopathy. A clue may be found in the LVEF, which is also enhanced in the 403 mice. These mice have impaired diastolic function, which is corrected in the G s x403 cross. It is our contention that by correcting the impaired diastolic relaxation, systolic function is permitted to increase further, which becomes even more noticeable in isolated myocytes. This concept is consistent with the point of view that the 403 mutation exhibits a gain of function, as has been suggested from single-molecule mechanics of whole cardiac myosin, 17 as well as pressure-volume relationships. 18 Thus, in the G s x403 model, the gain of function is augmented by correcting the diastolic dysfunction of 403 mice when mated with G s mice, which results in enhanced systolic function at baseline even in the absence of neurohormonal stimulation. This concept is supported by data with Rp-cAMP, a protein kinase A inhibitor, which did not affect baseline myocyte function. Thus, the elevated LV function and heart rate at baseline combined with augmented contractility on -adrenergic stimulation conceivably provide the framework for the accelerated cardiomyopathy observed in the present study. At least in this mouse model of FHC, there is no evidence that loss of function is the cause of cardiomyopathy. 19 As expected, protein levels of G s were increased similarly in G s and G s x403 compared with WT mice, but interestingly, G s -protein was downregulated in 403 mice. Because -adrenergic receptor density was unchanged in 403 mice, our findings suggest that in 403 mice, a 30% reduction in the protein level of G s may be responsible for the decreased responsiveness to -adrenergic stimulation in vivo and in isolated cardiomyocytes. However, it is just as conceivable that the correction of diastolic function and enhanced systolic function at baseline provide the framework for the augmented responses to -adrenergic stimulation, both at the level of isolated myocyte function and in vivo. Enhanced inotropic responses were not observed with the nonspecific inotropic agent Ca 2, which also confirms that enhanced -adrenergic signaling is critical to the development of the cardiomyopathy. Although we cannot entirely exclude the possibility that other G-protein coupled receptors or distal mechanisms may

7 620 Circulation February 5, 2002 play a role in the development of cardiomyopathy in G s x403 mice, our previous study demonstrated that the cardiomyopathy in G s mice was blocked completely by propranolol, 12 which further supports the concept that the cardiomyopathy in G s x403 mice is mediated through the -adrenergic signaling pathway. Summary The data from the present study show that the combination of overexpression of G s and the -myosin heavy chain mutation results in both accelerated and more severe cardiomyopathy than observed in either parent alone. It is likely that enhanced baseline LV function combined with elevated heart rate in young G s x403 mice accounts for the accelerated cardiomyopathy. These results provide a molecular underpinning to clinical studies, which have suggested that sympathetic stimulation may be deleterious both in the short and long term in patients with FHC and that -blocker therapy is beneficial in some patients with FHC. 20,21 Acknowledgments This study was supported in part by NIH HL-33107, HL-33065, HL-69020, HL-59139, HL-65182, HL-65183, and AG References 1. Thierfelder L, Watkins H, MacRae C, et al. Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell. 1994;77: Seidman JG, Seidman C. The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms. Cell. 2001;104: Geisterfer-Lowrance AA, Kass S, Tanigawa G, et al. A molecular basis for familial hypertrophic cardiomyopathy: a beta cardiac myosin heavy chain gene missense mutation. Cell. 1990;62: Bonne G, Carrier L, Richard P, et al. Familial hypertrophic cardiomyopathy: from mutations to functional defects. Circ Res. 1998;83: Doevendans PA. Hypertrophic cardiomyopathy: do we have the algorithm for life and death? Circulation. 2000;101: Marian AJ, Roberts R. Recent advances in the molecular genetics of hypertrophic cardiomyopathy. Circulation. 1995;92: Geisterfer-Lowrance AA, Christe M, Conner DA, et al. A mouse model of familial hypertrophic cardiomyopathy. Science. 1996;272: McConnell BK, Fatkin D, Semsarian C, et al. Comparison of two murine models of familial hypertrophic cardiomyopathy. Circ Res. 2001;88: Gaudin C, Ishikawa Y, Wight DC, et al. Overexpression of Gs alpha protein in the hearts of transgenic mice. J Clin Invest. 1995;95: Iwase M, Bishop SP, Uechi M, et al. Adverse effects of chronic endogenous sympathetic drive induced by cardiac Gs alpha overexpression. Circ Res. 1996;78: Iwase M, Uechi M, Vatner DE, et al. Cardiomyopathy induced by cardiac Gs alpha overexpression. Am J Physiol. 1997;272:H585 H Asai K, Yang GP, Geng YJ, et al. Beta-adrenergic receptor blockade arrests myocyte damage and preserves cardiac function in the transgenic Gs alpha mouse. J Clin Invest. 1999;104: Kim SJ, Iizuka K, Kelly RA, et al. An alpha-cardiac myosin heavy chain gene mutation impairs contraction and relaxation function of cardiac myocytes. Am J Physiol. 1999;276:H1780 H Kim SJ, Yatani A, Vatner DE, et al. Differential regulation of inotropy and lusitropy in overexpressed Gs alpha myocytes through camp and Ca2 channel pathways. J Clin Invest. 1999;103: Geng YJ, Ishikawa Y, Vatner DE, et al. Apoptosis of cardiac myocytes in Gs alpha transgenic mice. Circ Res. 1999;84: Vatner DE, Asai K, Iwase M, et al. Overexpression of myocardial Gs alpha prevents full expression of catecholamine desensitization despite increased beta-adrenergic receptor kinase. J Clin Invest. 1998;101: Tyska MJ, Hayes E, Giewat M, et al. Single-molecule mechanics of R403Q cardiac myosin isolated from the mouse model of familial hypertrophic cardiomyopathy. Circ Res. 2000;86: Georgakopoulos D, Christe ME, Giewat M, et al. The pathogenesis of familial hypertrophic cardiomyopathy: early and evolving effects from an alpha-cardiac myosin heavy chain missense mutation. Nat Med. 1999;5: Marian AJ. Pathogenesis of diverse clinical and pathological phenotypes in hypertrophic cardiomyopathy. Lancet. 2000;355: Loogen F, Kuhn H, Gietzen F, et al. Clinical course and prognosis of patients with typical and atypical hypertrophic obstructive and with hypertrophic non-obstructive cardiomyopathy. Eur Heart J. 1983;4(suppl F): Spirito P, Seidman CE, McKenna WJ, et al. The management of hypertrophic cardiomyopathy. N Engl J Med. 1997;336: