Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A

Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Atrial natriuretic peptide (ANP) has been postulated to exert local antihypertrophic effects in the heart. Thus, a loss of function of the ANP receptor guanylyl cyclase-A (GC-A) might contribute to the increased propensity to cardiac hypertrophy, although a causative role in vivo has not been definitively demonstrated. To test whether local ANP modulates cardiomyocyte growth, we inactivated the GC-A gene selectively in cardiomyocytes by homologous loxP/Cre-mediated recombination. Thereby we have circumvented the systemic, hypertensive phenotype associated with germline inactivation of GC-A. Mice with cardiomyocyte-restricted GC-A deletion exhibited mild cardiac hypertrophy, markedly increased mRNA expression of cardiac hypertrophy markers such as ANP (fivefold), α-skeletal actin (1.7-fold), and β-myosin heavy chain (twofold), and increased systemic circulating ANP levels. Their blood pressure was 7–10 mmHg below normal, probably because of the elevated systemic levels and endocrine actions of ANP. Furthermore, cardiac hypertrophic responses to aortic constriction were enhanced and accompanied by marked deterioration of cardiac function. This phenotype is consistent with a local function of the ANP/GC-A system to moderate the molecular program of cardiac hypertrophy.

The heart affects cardiovascular homeostasis by secreting two natriuretic peptides (NPs), atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) (1). They activate a common guanylyl cyclase-A (GC-A) receptor expressed in a variety of tissues, thereby increasing intracellular cGMP concentrations (2). NPs are secreted from atrial granules into the circulation in response to acute or chronic atrial stretch to act as antihypertensive and antihypervolemic factors through GC-A (3, 4). A second receptor subtype that also triggers the effects of NPs is the C-receptor (NP receptor-C, or NPR-C) (4), a clearance receptor that mediates the cellular internalization and degradation of NPs. It may also participate in mediating some of their cellular actions via coupling to G_i proteins and inhibition of adenylyl cyclase (5). However, within the cardiovascular system the primary action of the NPR-C seems to be the modulation of circulating and local NP concentrations (6).

In chronic hemodynamic overload, there is a significant increase in ANP and BNP expression in the cardiac ventricles (3). It is postulated that in this situation NPs exert not only endocrine but also local antihypertrophic actions (ANP) and antifibrotic actions (BNP) (7). For example, ANP inhibits growth and proliferation of cultured cardiac myocytes and fibroblasts via GC-A (8, 9). Also, overexpression of GC-A in cardiomyocytes exerts antihypertrophic effects in vivo (10). Conversely, mice with genetic disruption of the GC-A gene exhibit arterial hypertension and a marked cardiac hypertrophy that is disproportionate to their increased blood pressure and resistant to antihypertensive medication (11–13). By comparison, other mutant mice with similar increases in blood pressure do not have this pronounced hypertrophic phenotype (14). These observations suggest that the ANP/GC-A pathway could be involved in the regulation of myocyte size. However, efforts to prove this concept in vivo have been limited by the systemic, hypertensive phenotype of mice with global deletion of ANP or GC-A (11, 15).

To test whether ANP locally modulates cardiomyocyte growth and contractility, we generated mice with selective deletion of GC-A in cardiomyocytes. All endocrine blood pressure– and volume-regulating effects of ANP are preserved. Based on studies in these mice, we conclude that the ANP/GC-A system indeed plays a subtle but critical, autocrine/paracrine role in moderating cardiomyocyte growth and contractile function in vivo independently of its effects on blood pressure.

Cardiomyocyte-selective deletion of the floxed GC-A gene. Disruption of the GC-A gene in the hearts of CM GC-A KO mice was demonstrated by Southern blotting and RT-PCR. Cardiac DNA from flox/flox GC-A and CM GC-A KO mice was extracted, digested with BamHI, and hybridized to visualize the nonrecombined floxed allele and the recombined floxed-out allele. As shown in Figure 1, b and c, 23% ± 4% of the alleles in cardiac samples from CM GC-A KO mice have undergone recombination. Of note, entire cardiac tissue contains many other cells besides cardiomyocytes, such as vascular endothelial and smooth muscle cells and interstitial fibroblasts. All these cell types express GC-A (2, 4, 8), but only the GC-A gene expressed in cardiomyocytes undergoes αMHC-Cre-mediated recombination. In fact, only 14% of mouse ventricular cells are cardiomyocytes, and 90–95% of these are binucleated (23), indicating that 24% of ventricular DNA originates from cardiomyocytes. The recombination of about 20% of the alleles in hearts from CM GC-A KO mice indicates that about 80% of the cardiomyocytes have undergone recombination. This deletion event was never demonstrated in genomic tail DNA obtained from the respective CM GC-A KO mice (Figure 1b). In enriched cardiomyocytes, the ratio of recombined to nonrecombined alleles was 55% ± 3% (Figure 1, b and c). Since there is still substantial nonmyocyte contamination in this preparation, this number represents a lower estimate of the efficiency of Cre-mediated recombination in the cardiomyocyte population. Indeed, semiquantitative RT-PCR demonstrated that the expression of GC-A mRNA in single cardiomyocytes of CM GC-A KO mice is reduced by about 80% (Figure 2a). The CM GC-A KO mice grew to adulthood without signs of cardiac dysfunction and were grossly indistinguishable from their floxed GC-A littermates. Also, spontaneous lethality was not different between genotypes.

Figure 2

Demonstration of the GC-A deletion at the mRNA and protein level. (a) RT-PCR analyses of GC-A and GAPDH mRNA in preparations of isolated adult ventricular cardiomyocytes from floxed GC-A and CM GC-A KO mice (20–30 single cells were collected from each heart; five hearts were studied per genotype). Left: Ethidium bromide visualization of RT-PCR products. Left ventricle (LV) of a floxed GC-A mouse was used as positive control. Right: GC-A signal intensities were normalized to GAPDH. (b) Basal and ANP-stimulated guanylyl cyclase activity of plasma membranes isolated from whole hearts of floxed GC-A and CM GC-A KO mice. Enzymatic activity is expressed as picomoles of cGMP formed per milligram of protein over 10 minutes (n = 6). *P < 0.05 vs. floxed GC-A.

Decreased GC-A activity in cardiac membranes from CM GC-A KO mice. To further demonstrate the deletion of GC-A, membrane-bound guanylyl cyclase activity was assayed in hearts from floxed GC-A and CM GC-A KO mice. Figure 2b demonstrates that both basal and ANP-stimulated enzyme activities in CM GC-A KO hearts were significantly reduced, by approximately 18% and 30%, respectively. The high resting GC-A activity derives from vascular endothelial and smooth muscle cells as well as fibroblasts, which are all known to express GC-A and, as mentioned, constitute the predominant cell population in these whole-heart preparations.

CM GC-A KO mice exhibit decreased arterial blood pressure and cardiomyocyte hypertrophy. As shown in Figure 3a, systolic blood pressure of CM GC-A KO mice was slightly but significantly lower than that of age- and sex-matched floxed GC-A littermates, by about 7–10 mmHg (P < 0.01). In spite of this, there was a significant increase in the ratio of heart weight (HW) to body weight (BW) of CM GC-A KO versus floxed GC-A littermates (P = 0.012) (Figure 3b). As a result, the relationship between the left ventricular weight/body weight (LVW/BW) ratios and the systolic blood pressure levels differed significantly between genotypes (Figure 3c). Because the same relative changes were seen in both sexes, data from males and females were combined (n = 14 mice per sex and genotype).

Figure 3

Effect of cardiomyocyte-restricted disruption of the GC-A gene on systolic blood pressure (SBP) (a), heart weight/body weight (HW/BW) ratios (b), and the relationship between left ventricular weight/body weight (LVW/BW) ratios and SBP (c) (n = 14 per sex and genotype, n = 28 per group, *P < 0.05 vs. floxed GC-A).

Morphometrical analyses of PAS-stained sections demonstrated that cell diameters of ventricular cardiomyocytes from CM GC-A KO mice were significantly increased compared with those of their floxed GC-A littermates (15.44 ± 0.45 μm and 14.04 ± 0.3 μm, respectively; P = 0.018) (Figure 4, a and c). Ventricular collagen fractions, quantitated on Sirius red–stained sections, were similar in both genotypes (0.62% ± 0.03% in CM GC-A KO and 0.59% ± 0.02% in floxed GC-A mice) (Figure 4, b and d).

Figure 4

Histological analysis of floxed GC-A and CM GC-A KO hearts. Cardiomyocyte diameters and interstitial collagen fractions were estimated by quantitative morphometry of PAS-stained (a) or Sirius red–stained (b) ventricular sections, respectively. Black lines (a) indicate cardiomyocyte diameters in the region of the cell nucleus, which were significantly increased in CM GC-A KO hearts (c), without changes in interstitial collagen (d) (n = 16, *P < 0.05 vs. floxed GC-A).

Increased expression of cardiac hypertrophy genes in CM GC-A KO mice. Next, we examined ventricular mRNA expression of hypertrophy markers. Northern blot analyses revealed that CM GC-A KO mice exhibit increased ventricular expression levels of ANP, α-sk-actin, and βMHC (increased 4.9 ± 0.6–fold, 1.7 ± 0.2–fold, and 2 ± 0.6–fold, respectively; P < 0.05 vs. floxed GC-A) (Figure 5a). αMHC levels were not changed (Figure 5a). The βMHC/αMHC ratio was increased 2.7 ± 0.9–fold (n = 8, P < 0.05 vs. floxed GC-A).

Figure 5

Representative Northern blots. Messenger RNA expression levels of ANP, α-sk-actin, αMHC, and βMHC, as well as GAPDH, in cardiac ventricles of floxed GC-A and CM GC-A KO mice under baseline conditions (a) and after TAC (b).

CM GC-A KO mice exhibit increased circulating plasma ANP levels. To elucidate how the increased ventricular ANP mRNA levels in CM GC-A KO mice affect circulating concentrations, plasma levels were measured by RIA. As shown in Figure 6, plasma ANP levels were increased more than twofold in CM GC-A KO as compared with floxed GC-A littermates.

Figure 6

Plasma concentrations of ANP in CM GC-A KO mice compared with their floxed GC-A littermates (n = 18, *P < 0.05 vs. floxed GC-A).

CM GC-A KO mice show preserved cardiac contractility but decreased cardiac relaxation. Heart rate and left ventricular contractility (Figure 7, a–c) were similar in CM GC-A KO and floxed GC-A littermates (n = 6 per genotype, all 6–7 months old). Also, the chronotropic and inotropic responses to the β-agonist dobutamine were preserved (Figure 7, a–c). In contrast, the baseline maximal relaxation rates and the lusitropic responses of CM GC-A KO hearts to low levels of β-adrenergic stimulation were significantly decreased (Figure 7d).

Figure 7

In vivo hemodynamic measurements of cardiac function by left ventricular catheterization. Heart rate (a), peak left ventricular systolic pressure (LVSP) (b), and the maximal rates of contraction (dP/dt_max) (c) and relaxation (dP/dt_min) (d) are shown at baseline (0) and during infusion of dobutamine in CM GC-A KO and floxed GC-A mice (n = 6, *P < 0.05 vs. floxed GC-A).

Isolated cardiomyocytes from CM GC-A KO mice exhibit normal contractility. Because ventricular performance in vivo is a function of not only intrinsic myocyte contractility, but also many systemic parameters, the influence of GC-A ablation on cardiomyocyte contractility was also examined in isolated, electrically paced ventricular myocytes. Corroborating the histological data, videotaped images of single cells showed that the resting length and width of GC-A KO cardiomyocytes were significantly increased (Table 1). Baseline myocyte shortening was similar in both genotypes. ANP inhibited the systolic shortening of floxed GC-A cardiomyocytes by about 25%; in myocytes from CM GC-A KO mice, the effect of ANP was abolished. Isoproterenol increased cell shortening 2.7-fold in both genotypes (Table 1).

Table 1

Contractile parameters of isolated adult ventricular cardiomyocytes

CM GC-A KO mice have an enhanced cardiac hypertrophic response to pressure overload. To investigate the local role of ANP under conditions of pressure overload, mice from both genotypes were subjected to TAC. After 10 days of banding, the systolic blood pressure (proximal to the constriction) was lower in CM GC-A KO than in floxed GC-A mice (Figure 8a). Also, the induced pressure gradient was lower in CM GC-A KO mice (72 ± 3 mmHg vs. 92 ± 3.4 mmHg, P = 0.02). In spite of this, the average HW/BW and LVW/BW ratios were greater in banded CM GC-A KO than in floxed GC-A mice (HW/BW, 8.85 ± 0.14 vs. 7.67 ± 0.2, P = 0.002) (Figure 8b). Figure 8c shows the plotted LVW/\BW ratios of individual animals as a function of the systolic pressure proximal to the aortic constriction. For any total pressure load, the CM GC-A KO mice have an enhanced hypertrophic response (Figure 8d), suggesting that this enhancement is not simply a function of blood pressure. The mortality rates were similar in both genotypes.

Figure 8

(a) TAC led to a substantial increase in systolic blood pressure (SBP) proximal to the stenosis. SBP was significantly lower in the CM GC-A KO than in the floxed GC-A mice. For reference, pressures in sham-operated mice averaged 100 ± 6 mmHg (floxed GC-A) and 93 ± 10 mmHg (CM GC-A KO), indicating that a substantial pressure load was induced by TAC in both genotypes. (b) Despite the lower SBP, the CM GC-A KO mice had an increased LVW/BW index compared with the floxed GC-A mice. (c) The linear regression of SBP and LVW/BW index revealed that in the CM GC-A KO mice (filled circles and solid line), a stronger hypertrophic response in the heart correlated with lower blood pressures, while in the floxed GC-A mice (open circles and dashed line), blood pressure and LVW/BW index were positively correlated. (d) To compare the hypertrophic response independently of the differences in blood pressure in this figure, we divided the LVW/BW index of each animal by the individual SBP and normalized this value to the mean SBP of 145 mmHg (n = 18 floxed GC-A and 27 CM GC-A KO mice, *P < 0.05 vs. floxed GC-A).

Sirius red staining of left ventricular paraffin sections revealed that TAC caused a slight increase in interstitial collagen fractions — in floxed GC-A mice from 0.56 ± 0.03 (sham-operated) to 0.61 ± 0.05 (by 8%), and in CM GC-A KO mice from 0.57 ± 0.04 to 0.69 ± 0.05 (by 24%, P < 0.05). Thus, cardiac fibrosis in response to TAC was slightly enhanced in CM GC-A KO mice.

Left ventricles from aortic-banded floxed GC-A and CM GC-A KO mice were also analyzed for hypertrophic marker gene expression by Northern blotting. As compared with respective sham-operated mice, the increases in gene expression levels in floxed GC-A and CM GC-A KO mice were as follows: for ANP, 12.1- and 6.6-fold, respectively; for α-sk-actin, 2.6- and 1.9-fold, respectively; and for β/αMHC, 6.5- and 4.9-fold, respectively. As indicated in Figure 5b, the absolute ventricular mRNA levels of ANP and the ratios of βMHC to αMHC were significantly greater in banded CM GC-A KO than in floxed GC-A mice: 1.66 ± 0.1–fold and 1.72 ± 0.2–fold, respectively (n = 10, P < 0.05 vs. floxed GC-A).

CM GC-A KO mice have markedly impaired cardiac function after pressure overload. Last, given the lower arterial blood pressure observed in CM GC-A KO mice with TAC, we performed an additional series of experiments, using left ventricular catheterization to measure cardiac function in mice banded for 10–14 days. CM GC-A KO mice showed decreased left ventricular contractility and relaxation as compared with floxed GC-A littermates, as evidenced by a reduction of the maximal rate of left ventricular pressure increase (dP/dt_max) and decrease (dP/dt_min), as well as by an increased left ventricular end-diastolic pressure (Table 2). In addition, the ratios of lung weight (LW) and right ventricular weight (RVW) to BW were greater in TAC-operated CM GC-A KO than in floxed GC-A mice (LW/BW, 19.7 ± 1.5 vs. 13.8 ± 1.6, P < 0.05; RVW/BW, 2.0 ± 0.1 vs. 1.6 ± 0.1, P < 0.05 vs. floxed GC-A; n = 6), indicating congestive heart failure without clinical signs of right ventricular failure.

Table 2

Cardiac function after TAC

The present study describes a cardiomyocyte-restricted inactivation of the GC-A gene (CM GC-A KO) using the loxP/Cre system. Chronic abolition of the local, cardiac effects of ANP in mice resulted in a slight but significant increase in cardiomyocyte size in the absence of interstitial fibrosis, accompanied by a marked increased in the expression of cardiac hypertrophy markers such as ANP, α-sk-actin, and βMHC. Cardiac contractility was preserved, but relaxation was slightly impaired. Surprisingly, CM GC-A KO mice exhibit a mild but significant arterial hypotension by 7–10 mmHg, which is probably related to the increased levels of ANP in the systemic circulation and the subsequently enhanced hypotensive effects of the peptide. Furthermore, CM GC-A KO mice have an increased hypertrophic response to aortic constriction–induced pressure load, accompanied by a marked deterioration of cardiac function. Taken together, these results suggest that ANP has a subtle but critical GC-A–mediated function to moderate the molecular program of hypertrophy in the heart.

Under physiological conditions, ANP is synthesized and secreted mainly by atrial myocytes (1). However, during cardiac hypertrophy, i.e., in response to arterial hypertension, ventricular myocytes undergo phenotypic modifications that result in the re-expression of fetal genes, in particular ANP and BNP (3). It was postulated that in this situation local ANP could be directly involved in the regulation of cardiomyocyte growth (8–13). To dissect the local actions of ANP in vivo, the GC-A gene was deleted specifically in cardiomyocytes. Floxed GC-A mice (16) were mated with mice expressing Cre recombinase in cardiomyocytes (αMHC-Cre^tg) (17). Based on Southern blot and RT-PCR analysis of whole hearts and purified cardiomyocytes obtained from the resulting CM GC-A KO offspring, we estimate the efficacy of the cardiomyocyte GC-A gene deletion to be approximately 80%. Furthermore, ANP-stimulated guanylyl cyclase activity was markedly reduced in CM GC-A KO hearts, by about 30%; the high resting GC-A activity very likely derives from noncardiomyocyte cells in the heart. Indeed, the inhibitory effects of ANP on contractility of isolated cardiomyocytes in vitro are completely abolished in cardiac myocytes from CM GC-A KO mice, demonstrating the efficient deletion of GC-A.

Unexpectedly, loxP/Cre-mediated, cardiomyocyte-restricted deletion of GC-A resulted in mild arterial hypotension, and yet cardiac weight and cardiomyocyte size (measured by histological and video-edge detection analyses) were significantly increased. Cardiomyocyte hypertrophy was accompanied by enhanced ventricular expression of the hypertrophy markers ANP, α-sk-actin, and βMHC. In addition, CM GC-A KO mice have a greater cardiac hypertrophic response to TAC, which is accompanied by slightly enhanced cardiac fibrosis and marked impairment of cardiac function. These observations suggest that specific structural and/or molecular changes in GC-A–deficient cardiomyocytes compromise the adaptive responses to pressure overload. Taken together, our results suggest that ANP has a critical, GC-A–mediated local function to moderate cardiomyocyte growth under both normal and enhanced pressure-load conditions.

As demonstrated in vitro and in vivo, the resting contractile parameters of CM GC-A KO mice, and the inotropic responses of these mice to β-adrenergic stimulation, were preserved. Intriguingly, invasive hemodynamics revealed a slightly lower rate of cardiac relaxation both at baseline and under low levels of β-adrenergic stimulation. Interestingly, studies in humans (24) and rodents (25, 26) agree that ANP has little or no effect on cardiac contraction in vivo but directly accelerates relaxation. Deletion of cardiomyocyte GC-A may prevent this effect and thereby delay diastolic relaxation in CM GC-A KO mice. Obviously, impaired cardiac relaxation might also be related to specific structural and molecular changes within GC-A–deficient cardiomyocytes, an interesting question that we will address in future studies.

Since baseline cardiac contractility was normal and the mice clearly showed no clinical signs of cardiac dysfunction, we interpret the hypotensive effect of the cardiomyocyte-restricted ablation of GC-A as being caused by the markedly enhanced cardiac expression and secretion of ANP. Since the endocrine expression sites of GC-A (in the kidney, vasculature, adrenals, and CNS) are preserved, the increased circulating concentrations of ANP are likely to decrease blood pressure below normal. In this context the observation that CM GC-A KO mice exhibit increased cardiac expression and release of ANP is even more surprising, because the mild chronic arterial hypotension would be expected to induce, in turn, a homeostatic decrease in cardiac ANP secretion (3). In particular, two mechanisms might be responsible for this phenotype: (a) reactivation of a fetal gene expression program accompanying cardiomyocyte hypertrophy, and/or (b) abolition of an autoregulatory mechanism by which ANP inhibits its own secretion in an autocrine/paracrine GC-A/cGMP–mediated fashion (27). Indeed, cardiac overexpression of GC-A in mice drastically reduced ventricular ANP mRNA levels and concentrations (10). In the present study, the increases in ANP mRNA expression were disproportionally large compared with the increases in other hypertrophy marker genes, such as α-sk-actin and βMHC, suggesting that several mechanisms are involved.

Which cellular pathways are responsible for pressure-independent cardiac hypertrophy in CM GC-A KO mice? It is possible that high local levels of ANP and BNP could act via the other NP receptors expressed on cardiomyocytes, guanylyl cyclase-B (GC-B, a specific receptor for C-type natriuretic peptide) and NPR-C, to stimulate cardiomyocyte growth. However, the affinities of ANP and BNP are 100- to 1,000-fold lower for GC-B than for GC-A (28). Also, published studies in C-type natriuretic peptide (CNP) and NPR-C gene–disrupted mice found no evidence of altered cardiomyocyte growth (6, 29). Alternatively, one could postulate that high local ANP and BNP concentrations enhanced the release of growth-stimulating factors from neighboring fibroblasts. However, both peptides inhibit the proliferative and synthetic activities of cardiac fibroblasts instead of accentuating them (7, 8). Hence, it is unlikely that the increased expression and release of ANP contributed to the enhanced cardiac hypertrophic responses. Instead, the most likely explanation for our observations is that the inhibition of direct, GC-A–mediated effects of ANP caused the hypertrophic phenotype.

The molecular mechanism(s) by which ANP and GC-A inhibit cardiac hypertrophy is not definitively known. ANP inhibits MAPK activation via cGMP (30). Additionally, a recent study showed that cGMP-dependent protein kinase I inhibits calcineurin/NFAT–mediated cardiomyocyte hypertrophy (31). However, these observations were in cultured neonatal cardiomyocytes, which share many but not all of the properties of adult cells. In our future studies we will take advantage of this newly developed genetic mouse model to further characterize the specific molecular pathways that mediate the inhibitory effects of ANP in adult cardiac myocytes.

In patients with heart hypertrophy and/or heart insufficiency, the plasma levels of ANP are increased, but the vasodilative and natriuretic responses to the peptide are markedly attenuated, suggesting impaired receptor or postreceptor responsiveness of GC-A (32). It is unknown whether these changes also affect GC-A in the heart. Our studies in CM GC-A KO mice indicate that an inhibition of the local cardiac ANP effects would accelerate the progression of cardiac hypertrophy and insufficiency, and they support further work to assess the importance of the ANP/GC-A system — particularly its local cardiac alterations — in human cardiovascular diseases.

The authors thank Bernd Zetsche, Melanie Voß, and Dorothe Möllmann for excellent technical assistance. This work was supported by the Bundesministerium für Bildung und Forschung (BMBF 01EC9801), the University of Münster (Interdisziplinäre Klinische Forschung, IZKF B12), and the Deutsche Forschungsgemeinschaft (SFB 556) (all to M. Kuhn). M. van Eickels received an institutional grant from the University of Bonn (Programm zur gezielten Forschungsförderung an der medizinischen Fakultät [BONFOR]).

See the related Commentary beginning on page 1275.

Rita Holtwick and Martin van Eickels contributed equally to this work.

Martin van Eickels’ present address is: Medizinische Universitäts-Poliklinik, Bonn, Germany.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: natriuretic peptide (NP); atrial natriuretic peptide (ANP); B-type natriuretic peptide (BNP); guanylyl cyclase-A (GC-A); natriuretic peptide receptor-C (NPR-C); cardiomyocyte-restricted deletion of GC-A (CMGC-A KO); myosin heavy chain (MHC); periodic acid Schiff (PAS); α-skeletal actin (α-sk-actin); transverse aortic constriction (TAC); heart weight (HW); body weight (BW); left ventricular weight (LVW).

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