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Correlation of Myocyte Lengthening to Chamber Dilation in the Spontaneously Hypertensive Heart Failure (SHHF) Rat
J Mol Cell Cardiol 30, 2175–2181 (1998) Article No. mc980775
Correlation of Myocyte Lengthening to Chamber Dilation in the Spontaneously Hypertensive Heart Failure (SHHF) Rat Tetsutaro Tamura, Tatsuyuki Onodera, Suleman Said and A. Martin Gerdes Department of Anatomy and Structural Biology, University of South Dakota, School of Medicine, Vermillion, SD 57069, USA (Received 9 February 1998, accepted in revised form 14 July 1998) T. T, T. O, S. S A. M. G. Correlation of Myocyte Lengthening to Chamber Dilation in the Spontaneously Hypertensive Heart Failure (SHHF) Rat. Journal of Molecular and Cellular Cardiology (1998) 30, 2175–2181. Chronic congestive heart failure of various etiologies is characterized by progressive chamber dilation. Although myocyte lengthening is involved, it is not known if this cellular change can account for all of the chamber dilation. The controversy is due largely to technical limitations in collecting data on chamber circumference, myocyte length, and sarcomere length simultaneously. To address this issue, the contributions of myocyte and sarcomere lengthening to progressive chamber dilation in spontaneously hypertensive heart failure (SHHF) rats was examined using a new approach. Female SHHF rats (n=31) were examined at various time points between 2 months of age and the onset of end-stage heart failure (18 months or older). A new method enabled simultaneous collection of data on myocyte length, sarcomere length, and chamber circumference using formalin-fixed tissue. Reliability of cellular measurements was confirmed with an alternate method. LV myocyte length increased linearly between 2 and 24 months of age due to series addition of sarcomeres. Myocyte length increased in direct proportion to chamber circumference during this period (r=0.93, P<0.001). Results suggest that myocyte lengthening alone can account for chamber dilation in the progression to heart failure. Excessive myocyte lengthening is a slow, progressive change that begins long before clinical signs and symptoms of heart failure appear in this model of hypertension and failure. Since myocyte remodeling in hypertensive humans with and without failure is known to resemble that in SHHF rats, these data should provide important insight into chamber dilation and the progression of heart failure in humans. 1998 Academic Press
K W: Heart failure; Remodeling; Myocytes; Pathology.
Introduction Ventricular dilation is a well-known feature of chronic heart failure (Lasey et al., 1984; Vasan et al., 1997). It has been clearly established in humans and in animal models that myocyte lengthening, due to series addition of sarcomeres, is an important underlying feature of ventricular dilation in chronic heart failure (Zimmer et al., 1990; Gerdes et al., 1992, 1996; Gerdes, 1995). There is also a longstanding belief that myocyte slippage contributes to
chamber dilation in chronic heart failure (Linzbach, 1976; Olivetti et al., 1990; Beltrami et al., 1995). The dilemma in defining the relative contributions of myocyte slippage and lengthening to chamber dilation involves the technical difficulty in collecting myocyte length, sarcomere length, and chamber circumference simultaneously. A recently published, 11-year follow-up study of Framingham patients suggested that increased chamber diameter is a risk factor for the development of congestive heart failure (Vasan et al.,
Please address all correspondence to: A. Martin Gerdes, South Dakota Cardiovascular Research Institute, 1400 West 22nd Street, Sioux Falls, SD, 57105, USA.
0022–2828/98/112175+07 $30.00/0
1998 Academic Press
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1997). Though chamber dilation is clearly associated with chronic heart failure, temporal progression of dilation in individual patients has been difficult to determine due to the apparently slow progression of this change. Consequently, it may take many years to collect sufficient data on the cellular basis of progressive chamber dilation in humans. A new approach to isolate myocytes from formalin-fixed tissue for subsequent assessment of myocyte length and sarcomere length was implemented in our lab recently. The cell isolation procedure, which uses potassium hydroxide (KOH), was modified from a previous method outlined by Grabner and Pfitzer (1974). Since cell sizing is a new application for KOH-isolated myocytes, values for myocyte length and sarcomere length were validated by comparing to similar data collected from myocytes isolated using a standard collagenase aortic-perfusion method. Very little is known about the temporal cellular changes that underlie progressive chamber dilation leading to failure. Non-invasive methods, such as MRI and echocardiography, can provide information about chamber dimensions but there are no methods currently available to simultaneously define the cellular basis of these changes. In these experiments, temporal changes in myocyte length associated with chamber dilation in the progression to failure were examined in SHHF rats. Recentlypublished studies indicate that remodeling of cardiac myocyte shape in humans with and without heart failure and having a prior history of hypertension is identical to that in SHHF rats (Gerdes, 1995; Gerdes et al., 1996). Consequently, it is likely that the findings in this study are relevant to the human population.
fresh isolated myocytes using a standard collagenase technique (coronary perfusion). Collagenase digested cells were fixed in glutaraldehyde in 80 mmol/l phosphate buffer (Gerdes et al., 1986). All animals were treated in accordance with guidelines of the American Association for the Accreditation of Laboratory Animal Care.
Measurement of chamber circumference Rats were given an intraperitoneal injection of heparin (3000 U/kg, Sigma Chemical Company, St Louis, MO, USA) and euthanized in a 95% CO2 inhalant chamber. The hearts were quickly removed, trimmed, and rinsed in PBS (approximately 15°C) containing 137 mmol/l NaCl, 2.7 mmol/l KCl, 8.1 mmol/l Na2HPO4, and 1.5 mmol/l KH2PO4 (pH=7.3). Hearts were then weighed, and fixed immediately in 10% neutral buffered formalin (approximately 15°C). Approximately 2 weeks after immersion-fixation, each heart was sliced into 1mm-thick sections perpendicular to the base–apex axis approximately midway from the base to apex. The same section of each heart was used for the measurement of left-ventricular chamber circumference and isolation of myocytes using KOH. Mean chamber circumference was measured using video images of the tissue slice and a Jandel image analysis system (Jandel Scientific, San Rafael, CA, USA). The left-ventricular chamber circumference was defined as the average value of inner and outer circumference. Left-ventricular papillary muscles were ignored in determining inner chamber circumference.
Techniques for myocyte isolation
Materials and Methods Experimental model Lean female SHHF/Mcc-facp rats were obtained from Genetic Models Inc. (Indianapolis, IN, USA). SHHF indicates selective breeding for Spontaneous Hypertension and Heart Failure, and facp indicates that fa and cp obesity genes are allellic (McCune et al., 1995). Mcc indicates that the primary colony is maintained by Dr Sylvia McCune at Ohio State University. A total of 31 rats between 2 and 20 months of age were used to collect isolated myocytes with the KOH method. Twenty additional SHHF rats (age range 2–24 months) were used to prepare
Potassium hydroxide was use to prepare isolated myocytes from formalin-fixed tissue. After removing the right ventricular wall and left-ventricular papillary muscles, tissue was rinsed in PBS, cut into small pieces, and put into 12.5 mol/l KOH solution for 24 h. Subsequently, the pieces were transferred to PBS, vortexed vigorously for approximately 10 min, and poured through nylon mesh (250 lm). Freshly isolated cells were immediately fixed again in 10% buffered formaldehyde. Isolated cells were centrifuged through 3% Ficoll (Sigma Chemical Company) in 0.1 mol/l PBS to remove unwanted debris. Rod cells with normal sarcomere structure and no visible membrane damage (approximately 80% of cells) were judged as undamaged myocytes.
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Morphometry Cell length and sarcomere length were measured using a Jandel image analysis system (Jandel Scientific). Cell length, defined as the longest length parallel to the longitudinal axis, was measured in the first 50 undamaged single myocytes encountered in each prep using a 10× objective lens. For any direct comparisons of cell length between KOH-digested and collagenase-digested myocytes, KOH values were corrected to the resting, unloaded sarcomere length typical of collagenase-digested myocytes (e.g. 1.90 lm). The KOH procedure is not as effective as the standard collagenase method in isolating myocytes. Doublets and multiple cell clumps are more common. Consequently, measurements were collected only from myocytes with one or two nuclei. Myocytes with three or more nuclei, which comprised less than 2% of the population, were omitted. To visualize nuclei, myocytes were labeled with 4′-6′-diamino-2 phenylindole-2 HCl (DAPI). Sarcomere length was measured in the first 20 undamaged single myocytes encountered in each prep (10 sarcomeres per cell; 40× objective).
Statistical analysis Data were analysed using linear regression. Sampling size for myocyte length and sarcomere length from individual animals was sufficient to reduce sampling error to less than 5% in all animals (Rakusan et al., 1978). Summaries of individual data from animal groups (e.g. heart weight) are expressed as means±.. Summaries of mean data from animal groups (e.g. sarcomere length) are expressed as means±...
Results Lean female SHHF rats reached and maintained a fairly stable body mass by approximately four months of age (4 months, 206±9.4 g; 24 months, 229±19 g). Total heart weight, however, increased 108% during this period (4 months, 887±61 mg; 24 months, 1843±195 mg). Eight rats in the KOH group and four rats in the collagenase group
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Isolated myocytes were also prepared using a traditional method consisting of aortic perfusion with media followed by media plus collagenase. This method has been outlined in detail elsewhere (Gerdes et al., 1986).
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Figure 1 Linear regression analysis of age-related changes in myocyte length. Myocyte length continued to increase with aging in lean, female SHHF rats who maintain a relatively stable body mass after approximately 4 months of age. Normally, heart mass and myocyte dimensions do not change with aging in animals with stable body mass (indicated by dashed line; female Sprague–Dawley rats; Bai et al., 1990). Results were similar for collagenase-digested (Β; r=0.94; P<0.001) and KOH-digested myocytes (Χ; r=0.91; P<0.001). Cell length was adjusted to a sarcomere length of 1.90 lm in KOH-digested myocytes.
displayed signs of congestive heart failure (e.g. dyspnea, ascites, pleural effusion, pericardial effusion, cyanosis, etc.). As reported in our previous study (Gerdes et al., 1996), hemodynamics collected from animals with signs of heart failure also showed reduced LV peak systolic pressure and dP/dtmax (data not shown). The appearance of KOH-digested myocytes was similar to that of collagenase-digested cells. The depth of focus was considerably reduced in KOH cells, however, suggesting shrinkage in the minor transverse diameter. Striations, surface features, and nuclei (when stained with DAPI) are easily visualized in KOH-isolated myocytes. The percentage of cells with one, two, or more than two nuclei was 6.2, 92.2, and 1.6 respectively. Although an arresting agent was not employed, all KOH-digested preps displayed sarcomere lengths in the diastolic range of contraction. There were no significant differences in sarcomere lengths between KOH-digested preps from each time point (2 months, 2.07±0.06 lm; 6 months, 2.11±0.04 lm; 10 months, 2.00±0.05 lm; 18–20 months, 1.97±0.05 lm). Sarcomere length was 1.90±0.03 lm in collagenase-digested myocytes. In Figure 1, myocyte length for KOH and collagenase-digested cells was plotted v age after correcting KOH cell length values to a sarcomere
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Figure 2 Linear regression analysis of changes in cell length and chamber circumference in aging SHHF rats. Cell length increased linearly with chamber circumference (r=0.93; P<0.001; y=5.72x+22.1). There were no significant differences between the regression lines from non-failing (Β) and failing (Χ) rats, so data were pooled.
length of 1.90 lm. As indicated by the overlapping lines, both methods produced the same results. A progressive increase in cell length was observed as the animals age. The dotted line indicates that cell length does not change after 4 months of age in normal rats (Sprague–Dawley) who maintain a stable body mass (Bai et al., 1990). A significant linear correlation between cell length and chamber circumference is shown in Figure 2. Since regression lines for non-failing (open circles) and failing rats (closed circles) were not significantly different, data were combined. Although the regression lines were similar, heart failure was clearly associated with the largest values for myocyte length and chamber circumference. It can be seen from this figure that a two-fold increase in chamber circumference was associated with an approximate doubling of myocyte length. Since changes in cell length should be directly reflected by proportional alterations in chamber circumference, cell length data were not corrected for sarcomere length in this instance.
Discussion Data presented here demonstrate for the first time that increased cardiac myocyte length alone can account for the chamber dilation in hypertension progressing to failure. Additionally, it was shown that cell lengthening is a slow, progressive, linear process that begins long before the development of clinical signs of failure in SHHF rats. The increase in cell length was due to an increase in the number
of series sarcomeres. Calculations from data presented here indicate that approximately 1.5 new sarcomeres per month were added to LV myocytes between 4 and 24 months of age. Although myocyte lengthening was linearly associated with aging, it is unlikely that this cellular process was due to aging, rather than hypertensive heart disease, since it is known that cardiac mass and myocyte dimensions do not change during aging in normal animals with stable body mass (e.g. mean LV myocyte length was 124, 124, and 126 lm in 4-, 8-, and 24-month-old female Sprague–Dawley rats, respectively; Bai et al., 1990). Cell length/chamber circumference alterations progressed in a similar manner after the onset of overt signs of congestive heart failure. Consequently, it appears that the rate of myocyte lengthening continued in a linear manner rather than becoming more accelerated with the transition to clinically detectable heart failure. Although these results are open to various interpretations, we believe that progressive myocyte lengthening eventually overwhelms compensatory neurohumoral mechanisms and overt failure ensues. It is known that increased myocyte length, due to series addition of sarcomeres, is a major underlying feature of chamber dilation in patients with heart failure due to ischemic and dilated cardiomyopathy (Gerdes et al., 1992, 1995; Gerdes, 1995; Zafeiridis et al., 1998). Additionally, it was shown recently that LV myocyte remodeling in SHHF rats progressing to failure was identical to that observed in human hypertensives progressing to failure (Gerdes, 1995; Gerdes et al., 1996; summarized in Fig. 3). Thus, the SHHF rat model should be useful in understanding the cellular and molecular mechanisms of altered myocyte shape in patients who develop chronic heart failure due to hypertension. Data submitted here suggest that maladaptive myocyte remodeling can begin long before the onset of overt signs of heart failure. Although recent data from cardiac explants obtained at the time of transplantation have demonstrated clear endpoints, we have no information regarding the temporal nature of myocyte remodeling in humans with cardiac hypertrophy progressing to failure. Based on data provided here, it is likely that maladaptive remodeling of cardiac myocyte shape may also develop and progress slowly in patients with hypertension. Indeed, if a similar time-frame occurs in humans (e.g. 40% cell lengthening over three quarters of the individuals life span), this may provide an explanation for the difficulty cardiologists have had in defining temporal chamber dilation in patients progressing towards failure
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Figure 3 Comparison of left-ventricular myocyte dimensions in humans with a prior history of hypertension and SHHF rats. Values for myocyte dimensions are similar in humans and rats with and without congestive heart failure. Data are summarized from Gerdes et al. (Gerdes et al., 1992, 1995; Gerdes, 1995). ∗ indicates P<0.01 for comparisons between failing and non-failing individuals within the rat or human groups; closed bars, hypertension/non-failing; open bars, heart failure with prior hypertension; n=8 for both rat groups; n=3 for hypertension/non-failing humans and n=4 for hypertension/failing humans.
(Vasan et al., 1997). The new technique used here to collect myocyte length, sarcomere length, and chamber circumference from formalin-fixed tissue may enable similar information to be collected from human autopsy or, perhaps, biopsy material in the future. Unfortunately, it will likely take many years before similar temporal data from a human population are collected. It should be noted that a 15% increase in left-ventricular myocyte length (e.g. about 10 sarcomeres) was noted in adjacent surviving myocardium 1 month after infarction in rats (Zimmer et al., 1990). Consequently, the time frame for myocyte remodeling may be more accelerated in other types of heart disease.
Potential limitations of the study Interpretation of the results in this study would have been complicated by the presence of large areas of myocardial scarring. Fortunately, little myocardial fibrosis was present in these lean female SHHF rats. Preliminary analysis of myocardial collagen content in lean female SHHF rats shows normal content at 12 months of age, but an increase from 1.9 to 2.5% between 12 and 24 months of age (unpublished observation). Thus, lean female SHHF rats develop a relatively small though significant amount of myocardial fibrosis with progression to failure. We have recently noted a much greater increase in myocardial collagen content in
male SHHF rats (unpublished observations). Consequently, it appears that, as in SHR rats (Pfeffer et al., 1982), gender-related differences in myocardial collagen content are also present in SHHF rats. How this relates to the accelerated progression to failure in male v female SHHF rats merits further study. Regarding the current study, it is unlikely that myocyte data collected here were adversely affected by the relatively small increase in myocardial fibrosis (e.g. there were no large areas of scarring that would have been difficult to interpret in the context of this study). It is known that the heart tends to become more globular in shape with the development of dilated heart failure (Cohn, 1995; Litwin et al., 1995). Potential regional variation due to the effects of this progressive geometric change in the heart were not addressed in the current study. Since such adjustments would have added considerable complexity to the study, we elected to examine only the middle portion of the heart. Although we believe that similar conclusions would have been reached by examining other regions of the heart, it is realized that this can not be stated with certainty in the absence of supportive data. A number of approaches were examined in pilot studies to address the potential problem of distinguishing individual myocytes from doublets or multicellular clumps in the KOH-digested preps. It was concluded that the most efficient and consistent manner to deal with this problem was to eliminate
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any myocytes containing three or more nuclei. Since these cells comprised less than 2% of the population of rat left-ventricular myocytes, any resulting error in measurements would be negligible. It should also be noted that observers could usually recognize intercalated discs between multiple cells. An additional aid in recognition of multiple cells was the irregular, stair-stepped transition from one cell to the next at intercalated discs. It has been suggested that “myocyte slippage” plays a major role in progressive chamber dilation leading to failure (Linzbach, 1976; Olivetti et al., 1990) though much of the literature mentioning this phenomenon is rather vague. This concept usually refers to slippage of myocytes past one another transversely or linear slippage of individual myofibrils within myocytes (Linzbach, 1976; Komamura et al., 1993). It would seem more likely that linear slippage of myocytes past one another could occur only at fascial planes due to the complex interdigitating connections between myocytes and the presence of intermyocyte collagen struts (Hoyt et al., 1989). Unfortunately, it is difficult to quantitate myocyte slippage directly. Linzbach (1976) and others (Olivetti et al., 1990) have noted a reduction in the number of myocytes across the wall as evidence of myocyte slippage. In our hands, we have found this approach to be subjective and inconsistent. Loss of Z-line registration has also been cited as evidence supporting myocyte slippage in various models of cardiac hypertrophy and failure (Ross et al., 1971; Papadimitriou et al., 1974; Hatt et al., 1979; Komamura et al., 1993; Sharov et al., 1994). While the evidence for this phenomenon appears to be strong, it should be noted that variation in sectioning angle or non-uniform fixation of sarcomeres can produce similar changes. In our experience, we have not observed a significant loss of sarcomere registration in any model of hypertrophy or failure examined to date. This includes the recent examination of six perfusion-fixed hearts obtained from SHHF rats with heart failure (unpublished observation). This study clearly demonstrates that myocyte length and sarcomere length can account for changes in chamber circumference leading to heart failure from hypertension. Although data submitted here do not completely exclude the possibility of myocyte slippage in this model, it appears that any such contributions are relatively insignificant and would be very difficult to detect. It should be realized that fiber direction changes across the wall much like a Japanese fan (Streeter and Hanna, 1973; Pearlman et al., 1982). While
the mid-myocardial portion of the ventricular wall (approximately half of the wall thickness) generally runs in a circumferential direction within transverse slices, measurements were also collected from epicardial and endocardial cells arranged obliquely or perpendicular to the plane of reference. If significant transmural differences in myocyte length were present across the wall, measurements could have been adversely affected. This is a very unlikely possibility, however, since we have not observed significant transmural differences in myocyte length in any of the models of cardiac hypertrophy examined in our lab over the years.
Acknowledgements This work was supported by grant HL 30696 from the National Institutes of Health. We are grateful to Dr Scott Campbell, Carrie Kline and Misty Smith for technical assistance.
References B S, C SE, M JA, M MC, G AM, 1990. Influence of aging, growth, and sex on cardiac myocyte size and number. Anat Rec 226: 207– 212. B CA, F N, R M, F GA, P C, C E, S EH, O G, A P, 1995. The cellular basis of dilated cardiomyopathy in humans. J Mol Cell Cardiol 27: 291–305. C J, 1995. Critical review of heart failure: the role of left ventricular remodeling in the therapeutic response. Clin Cardiol 18: IV4–IV12. G AM, 1995. Chronic ischemic heart disease. In: Weber KT (ed.). Wound Healing in Cardiovascular Disease. Armonk, NY: Futura Publishing Co., 61–66. G AM, M JA, H JM, K PA, B SP, 1986. Regional differences in myocyte size in normal rat heart. Anat Rec 215: 420–426. G AM, K SE, M JA, M KE, C LC, R PY, M KB, MK PP, S DD, 1992. Structural remodeling of cardiac myocytes in patients with ischemic cardiomyopathy. Circulation 86: 426–430. G AM, K SE, S DD, 1995. Implications of cardiomyocyte remodeling in heart dysfunction. In: Dhalla NS, Beamish RE, Takeda N and Nagano M (eds). The Failing Heart, NY. New York: Raven Press, 197–205. G AM, O T, W X, MC SA, 1996. Myocyte remodeling during the progression to failure in rats with hypertension. Hypertension 28: 609–614. G W, P P, 1974. Number of nuclei in isolated myocardial cells of pigs. Virchows Arch Abt B 15: 279–294. H PY, R K, G P, LP M, 1979. Morphometry and ultrastructure of heart hypertrophy
Myocyte Remodeling in Heart Failure induced by chronic volume overload (aorto-caval fistula in the rat). J Mol Cell Cardiol 11: 989–998. H RH, C ML, S JE, 1989. Distribution and three-dimensional structure of intercellular junctions in canine myocardium. Circ Res 64: 563–574. K K, S RP, I T, S YT, M I, B SP, V SF, 1993. Exhaustion of Frank– Starling mechanism in conscious dogs with heart failure. Am J Physiol 265: H1119–H1131. L WK, S MSJ, Z G, H JW, R N, 1984. Left ventricular mechanics in dilated cardiomyopathy. Am J Cardiol 54: 620–625. L AJ, 1976. Hypertrophy, hyperplasia and structural dilation of the human heart. Adv Cardiol 18: 1–14. L SE, K SE, W EO, L BH, A GP, D PS, 1995. Serial echocardiographic-doppler assessment of left ventricular geometry and function in rats with pressure-overload hypertrophy: chronic angiotensin-converting enzyme inhibition attenuates the transition to heart failure. Circulation 91: 2642–2654. MC SA, P S, R MJ, J RR, 1995. The SHHF/Mcc-facp rat model: a genetic model of congestive heart failure. In: Singal PK, Beamish RE and Dhalla NS (eds). Subcellular Basis and Therapy of Heart Failure. Boston, MA: Kluwer Academic Publishers, 91–106. O G, C JM, S EH, A P, 1990. Side-to-side slippage of myocytes participates in ventricular wall remodeling acutely after myocardial infarction in rats. Circ Res 67: 23–34. P JM, H BE, T RR, 1974. Regression of left ventricular dilation and hypertrophy after removal of volume overload: morphological and ultrastructural study. Circ Res 35: 127–135.
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P ES, W KT, J JS, P GG, F AP, 1982. Muscle fiber orientation and connective tissue content in the hypertrophied human heart. Lab Invest 46: 158–164. P JM, P MA, F P, F MC, B E, 1982. Favorable effects of therapy on cardiac performance in spontaneously hypertensive rats. Am J Physiol 242: H776–H784. R K, R S, L R, K B, 1978. The influence of aging and growth on the postnatal development of cardiac muscle in rats. Circ Res 42: 212–218. R J, S EH, T RR, S HM, C JW, 1971. Diastolic geometry and sarcomere lengths in the chronically dilated canine left ventricle. Circ Res 28: 49–61. S VG, S HN, S H, A AS, L TB, L M, G S, 1994. Abnormalities of contractile structures in viable myocytes of the failing heart. Int J Cardiol 43: 287–297. S DD, H WT, 1973. Engineering mechanics for successive states in canine left ventricular myocardium: fiber angle and sarcomere length. Circ Res 33: 656–664. V RS, L MG, B EJ, E JC, L D, 1997. Left ventricular dilatation and the risk of congestive heart failure in people without myocardial infarction. N Engl J Med 336: 1350–1355. Z A, J V, H SR, M KB, 1998. Regression of cellular hypertrophy following left ventricular assist device support. Circulation 98: 656–662. Z HG, G AM, M G, 1990. Changes in heart function and cardiac cell shape in rats with chronic myocardial infarction. J Mol Cell Cardiol 22: 1231–1243.
Correlation of Myocyte Lengthening to Chamber Dilation in the Spontaneously Hypertensive Heart Failure (SHHF) Rat Tetsutaro Tamura, Tatsuyuki Onodera, Suleman Said and A. Martin Gerdes Department of Anatomy and Structural Biology, University of South Dakota, School of Medicine, Vermillion, SD 57069, USA (Received 9 February 1998, accepted in revised form 14 July 1998) T. T, T. O, S. S A. M. G. Correlation of Myocyte Lengthening to Chamber Dilation in the Spontaneously Hypertensive Heart Failure (SHHF) Rat. Journal of Molecular and Cellular Cardiology (1998) 30, 2175–2181. Chronic congestive heart failure of various etiologies is characterized by progressive chamber dilation. Although myocyte lengthening is involved, it is not known if this cellular change can account for all of the chamber dilation. The controversy is due largely to technical limitations in collecting data on chamber circumference, myocyte length, and sarcomere length simultaneously. To address this issue, the contributions of myocyte and sarcomere lengthening to progressive chamber dilation in spontaneously hypertensive heart failure (SHHF) rats was examined using a new approach. Female SHHF rats (n=31) were examined at various time points between 2 months of age and the onset of end-stage heart failure (18 months or older). A new method enabled simultaneous collection of data on myocyte length, sarcomere length, and chamber circumference using formalin-fixed tissue. Reliability of cellular measurements was confirmed with an alternate method. LV myocyte length increased linearly between 2 and 24 months of age due to series addition of sarcomeres. Myocyte length increased in direct proportion to chamber circumference during this period (r=0.93, P<0.001). Results suggest that myocyte lengthening alone can account for chamber dilation in the progression to heart failure. Excessive myocyte lengthening is a slow, progressive change that begins long before clinical signs and symptoms of heart failure appear in this model of hypertension and failure. Since myocyte remodeling in hypertensive humans with and without failure is known to resemble that in SHHF rats, these data should provide important insight into chamber dilation and the progression of heart failure in humans. 1998 Academic Press
K W: Heart failure; Remodeling; Myocytes; Pathology.
Introduction Ventricular dilation is a well-known feature of chronic heart failure (Lasey et al., 1984; Vasan et al., 1997). It has been clearly established in humans and in animal models that myocyte lengthening, due to series addition of sarcomeres, is an important underlying feature of ventricular dilation in chronic heart failure (Zimmer et al., 1990; Gerdes et al., 1992, 1996; Gerdes, 1995). There is also a longstanding belief that myocyte slippage contributes to
chamber dilation in chronic heart failure (Linzbach, 1976; Olivetti et al., 1990; Beltrami et al., 1995). The dilemma in defining the relative contributions of myocyte slippage and lengthening to chamber dilation involves the technical difficulty in collecting myocyte length, sarcomere length, and chamber circumference simultaneously. A recently published, 11-year follow-up study of Framingham patients suggested that increased chamber diameter is a risk factor for the development of congestive heart failure (Vasan et al.,
Please address all correspondence to: A. Martin Gerdes, South Dakota Cardiovascular Research Institute, 1400 West 22nd Street, Sioux Falls, SD, 57105, USA.
0022–2828/98/112175+07 $30.00/0
1998 Academic Press
2176
T. Tamura et al.
1997). Though chamber dilation is clearly associated with chronic heart failure, temporal progression of dilation in individual patients has been difficult to determine due to the apparently slow progression of this change. Consequently, it may take many years to collect sufficient data on the cellular basis of progressive chamber dilation in humans. A new approach to isolate myocytes from formalin-fixed tissue for subsequent assessment of myocyte length and sarcomere length was implemented in our lab recently. The cell isolation procedure, which uses potassium hydroxide (KOH), was modified from a previous method outlined by Grabner and Pfitzer (1974). Since cell sizing is a new application for KOH-isolated myocytes, values for myocyte length and sarcomere length were validated by comparing to similar data collected from myocytes isolated using a standard collagenase aortic-perfusion method. Very little is known about the temporal cellular changes that underlie progressive chamber dilation leading to failure. Non-invasive methods, such as MRI and echocardiography, can provide information about chamber dimensions but there are no methods currently available to simultaneously define the cellular basis of these changes. In these experiments, temporal changes in myocyte length associated with chamber dilation in the progression to failure were examined in SHHF rats. Recentlypublished studies indicate that remodeling of cardiac myocyte shape in humans with and without heart failure and having a prior history of hypertension is identical to that in SHHF rats (Gerdes, 1995; Gerdes et al., 1996). Consequently, it is likely that the findings in this study are relevant to the human population.
fresh isolated myocytes using a standard collagenase technique (coronary perfusion). Collagenase digested cells were fixed in glutaraldehyde in 80 mmol/l phosphate buffer (Gerdes et al., 1986). All animals were treated in accordance with guidelines of the American Association for the Accreditation of Laboratory Animal Care.
Measurement of chamber circumference Rats were given an intraperitoneal injection of heparin (3000 U/kg, Sigma Chemical Company, St Louis, MO, USA) and euthanized in a 95% CO2 inhalant chamber. The hearts were quickly removed, trimmed, and rinsed in PBS (approximately 15°C) containing 137 mmol/l NaCl, 2.7 mmol/l KCl, 8.1 mmol/l Na2HPO4, and 1.5 mmol/l KH2PO4 (pH=7.3). Hearts were then weighed, and fixed immediately in 10% neutral buffered formalin (approximately 15°C). Approximately 2 weeks after immersion-fixation, each heart was sliced into 1mm-thick sections perpendicular to the base–apex axis approximately midway from the base to apex. The same section of each heart was used for the measurement of left-ventricular chamber circumference and isolation of myocytes using KOH. Mean chamber circumference was measured using video images of the tissue slice and a Jandel image analysis system (Jandel Scientific, San Rafael, CA, USA). The left-ventricular chamber circumference was defined as the average value of inner and outer circumference. Left-ventricular papillary muscles were ignored in determining inner chamber circumference.
Techniques for myocyte isolation
Materials and Methods Experimental model Lean female SHHF/Mcc-facp rats were obtained from Genetic Models Inc. (Indianapolis, IN, USA). SHHF indicates selective breeding for Spontaneous Hypertension and Heart Failure, and facp indicates that fa and cp obesity genes are allellic (McCune et al., 1995). Mcc indicates that the primary colony is maintained by Dr Sylvia McCune at Ohio State University. A total of 31 rats between 2 and 20 months of age were used to collect isolated myocytes with the KOH method. Twenty additional SHHF rats (age range 2–24 months) were used to prepare
Potassium hydroxide was use to prepare isolated myocytes from formalin-fixed tissue. After removing the right ventricular wall and left-ventricular papillary muscles, tissue was rinsed in PBS, cut into small pieces, and put into 12.5 mol/l KOH solution for 24 h. Subsequently, the pieces were transferred to PBS, vortexed vigorously for approximately 10 min, and poured through nylon mesh (250 lm). Freshly isolated cells were immediately fixed again in 10% buffered formaldehyde. Isolated cells were centrifuged through 3% Ficoll (Sigma Chemical Company) in 0.1 mol/l PBS to remove unwanted debris. Rod cells with normal sarcomere structure and no visible membrane damage (approximately 80% of cells) were judged as undamaged myocytes.
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Morphometry Cell length and sarcomere length were measured using a Jandel image analysis system (Jandel Scientific). Cell length, defined as the longest length parallel to the longitudinal axis, was measured in the first 50 undamaged single myocytes encountered in each prep using a 10× objective lens. For any direct comparisons of cell length between KOH-digested and collagenase-digested myocytes, KOH values were corrected to the resting, unloaded sarcomere length typical of collagenase-digested myocytes (e.g. 1.90 lm). The KOH procedure is not as effective as the standard collagenase method in isolating myocytes. Doublets and multiple cell clumps are more common. Consequently, measurements were collected only from myocytes with one or two nuclei. Myocytes with three or more nuclei, which comprised less than 2% of the population, were omitted. To visualize nuclei, myocytes were labeled with 4′-6′-diamino-2 phenylindole-2 HCl (DAPI). Sarcomere length was measured in the first 20 undamaged single myocytes encountered in each prep (10 sarcomeres per cell; 40× objective).
Statistical analysis Data were analysed using linear regression. Sampling size for myocyte length and sarcomere length from individual animals was sufficient to reduce sampling error to less than 5% in all animals (Rakusan et al., 1978). Summaries of individual data from animal groups (e.g. heart weight) are expressed as means±.. Summaries of mean data from animal groups (e.g. sarcomere length) are expressed as means±...
Results Lean female SHHF rats reached and maintained a fairly stable body mass by approximately four months of age (4 months, 206±9.4 g; 24 months, 229±19 g). Total heart weight, however, increased 108% during this period (4 months, 887±61 mg; 24 months, 1843±195 mg). Eight rats in the KOH group and four rats in the collagenase group
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Isolated myocytes were also prepared using a traditional method consisting of aortic perfusion with media followed by media plus collagenase. This method has been outlined in detail elsewhere (Gerdes et al., 1986).
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Figure 1 Linear regression analysis of age-related changes in myocyte length. Myocyte length continued to increase with aging in lean, female SHHF rats who maintain a relatively stable body mass after approximately 4 months of age. Normally, heart mass and myocyte dimensions do not change with aging in animals with stable body mass (indicated by dashed line; female Sprague–Dawley rats; Bai et al., 1990). Results were similar for collagenase-digested (Β; r=0.94; P<0.001) and KOH-digested myocytes (Χ; r=0.91; P<0.001). Cell length was adjusted to a sarcomere length of 1.90 lm in KOH-digested myocytes.
displayed signs of congestive heart failure (e.g. dyspnea, ascites, pleural effusion, pericardial effusion, cyanosis, etc.). As reported in our previous study (Gerdes et al., 1996), hemodynamics collected from animals with signs of heart failure also showed reduced LV peak systolic pressure and dP/dtmax (data not shown). The appearance of KOH-digested myocytes was similar to that of collagenase-digested cells. The depth of focus was considerably reduced in KOH cells, however, suggesting shrinkage in the minor transverse diameter. Striations, surface features, and nuclei (when stained with DAPI) are easily visualized in KOH-isolated myocytes. The percentage of cells with one, two, or more than two nuclei was 6.2, 92.2, and 1.6 respectively. Although an arresting agent was not employed, all KOH-digested preps displayed sarcomere lengths in the diastolic range of contraction. There were no significant differences in sarcomere lengths between KOH-digested preps from each time point (2 months, 2.07±0.06 lm; 6 months, 2.11±0.04 lm; 10 months, 2.00±0.05 lm; 18–20 months, 1.97±0.05 lm). Sarcomere length was 1.90±0.03 lm in collagenase-digested myocytes. In Figure 1, myocyte length for KOH and collagenase-digested cells was plotted v age after correcting KOH cell length values to a sarcomere
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Figure 2 Linear regression analysis of changes in cell length and chamber circumference in aging SHHF rats. Cell length increased linearly with chamber circumference (r=0.93; P<0.001; y=5.72x+22.1). There were no significant differences between the regression lines from non-failing (Β) and failing (Χ) rats, so data were pooled.
length of 1.90 lm. As indicated by the overlapping lines, both methods produced the same results. A progressive increase in cell length was observed as the animals age. The dotted line indicates that cell length does not change after 4 months of age in normal rats (Sprague–Dawley) who maintain a stable body mass (Bai et al., 1990). A significant linear correlation between cell length and chamber circumference is shown in Figure 2. Since regression lines for non-failing (open circles) and failing rats (closed circles) were not significantly different, data were combined. Although the regression lines were similar, heart failure was clearly associated with the largest values for myocyte length and chamber circumference. It can be seen from this figure that a two-fold increase in chamber circumference was associated with an approximate doubling of myocyte length. Since changes in cell length should be directly reflected by proportional alterations in chamber circumference, cell length data were not corrected for sarcomere length in this instance.
Discussion Data presented here demonstrate for the first time that increased cardiac myocyte length alone can account for the chamber dilation in hypertension progressing to failure. Additionally, it was shown that cell lengthening is a slow, progressive, linear process that begins long before the development of clinical signs of failure in SHHF rats. The increase in cell length was due to an increase in the number
of series sarcomeres. Calculations from data presented here indicate that approximately 1.5 new sarcomeres per month were added to LV myocytes between 4 and 24 months of age. Although myocyte lengthening was linearly associated with aging, it is unlikely that this cellular process was due to aging, rather than hypertensive heart disease, since it is known that cardiac mass and myocyte dimensions do not change during aging in normal animals with stable body mass (e.g. mean LV myocyte length was 124, 124, and 126 lm in 4-, 8-, and 24-month-old female Sprague–Dawley rats, respectively; Bai et al., 1990). Cell length/chamber circumference alterations progressed in a similar manner after the onset of overt signs of congestive heart failure. Consequently, it appears that the rate of myocyte lengthening continued in a linear manner rather than becoming more accelerated with the transition to clinically detectable heart failure. Although these results are open to various interpretations, we believe that progressive myocyte lengthening eventually overwhelms compensatory neurohumoral mechanisms and overt failure ensues. It is known that increased myocyte length, due to series addition of sarcomeres, is a major underlying feature of chamber dilation in patients with heart failure due to ischemic and dilated cardiomyopathy (Gerdes et al., 1992, 1995; Gerdes, 1995; Zafeiridis et al., 1998). Additionally, it was shown recently that LV myocyte remodeling in SHHF rats progressing to failure was identical to that observed in human hypertensives progressing to failure (Gerdes, 1995; Gerdes et al., 1996; summarized in Fig. 3). Thus, the SHHF rat model should be useful in understanding the cellular and molecular mechanisms of altered myocyte shape in patients who develop chronic heart failure due to hypertension. Data submitted here suggest that maladaptive myocyte remodeling can begin long before the onset of overt signs of heart failure. Although recent data from cardiac explants obtained at the time of transplantation have demonstrated clear endpoints, we have no information regarding the temporal nature of myocyte remodeling in humans with cardiac hypertrophy progressing to failure. Based on data provided here, it is likely that maladaptive remodeling of cardiac myocyte shape may also develop and progress slowly in patients with hypertension. Indeed, if a similar time-frame occurs in humans (e.g. 40% cell lengthening over three quarters of the individuals life span), this may provide an explanation for the difficulty cardiologists have had in defining temporal chamber dilation in patients progressing towards failure
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Figure 3 Comparison of left-ventricular myocyte dimensions in humans with a prior history of hypertension and SHHF rats. Values for myocyte dimensions are similar in humans and rats with and without congestive heart failure. Data are summarized from Gerdes et al. (Gerdes et al., 1992, 1995; Gerdes, 1995). ∗ indicates P<0.01 for comparisons between failing and non-failing individuals within the rat or human groups; closed bars, hypertension/non-failing; open bars, heart failure with prior hypertension; n=8 for both rat groups; n=3 for hypertension/non-failing humans and n=4 for hypertension/failing humans.
(Vasan et al., 1997). The new technique used here to collect myocyte length, sarcomere length, and chamber circumference from formalin-fixed tissue may enable similar information to be collected from human autopsy or, perhaps, biopsy material in the future. Unfortunately, it will likely take many years before similar temporal data from a human population are collected. It should be noted that a 15% increase in left-ventricular myocyte length (e.g. about 10 sarcomeres) was noted in adjacent surviving myocardium 1 month after infarction in rats (Zimmer et al., 1990). Consequently, the time frame for myocyte remodeling may be more accelerated in other types of heart disease.
Potential limitations of the study Interpretation of the results in this study would have been complicated by the presence of large areas of myocardial scarring. Fortunately, little myocardial fibrosis was present in these lean female SHHF rats. Preliminary analysis of myocardial collagen content in lean female SHHF rats shows normal content at 12 months of age, but an increase from 1.9 to 2.5% between 12 and 24 months of age (unpublished observation). Thus, lean female SHHF rats develop a relatively small though significant amount of myocardial fibrosis with progression to failure. We have recently noted a much greater increase in myocardial collagen content in
male SHHF rats (unpublished observations). Consequently, it appears that, as in SHR rats (Pfeffer et al., 1982), gender-related differences in myocardial collagen content are also present in SHHF rats. How this relates to the accelerated progression to failure in male v female SHHF rats merits further study. Regarding the current study, it is unlikely that myocyte data collected here were adversely affected by the relatively small increase in myocardial fibrosis (e.g. there were no large areas of scarring that would have been difficult to interpret in the context of this study). It is known that the heart tends to become more globular in shape with the development of dilated heart failure (Cohn, 1995; Litwin et al., 1995). Potential regional variation due to the effects of this progressive geometric change in the heart were not addressed in the current study. Since such adjustments would have added considerable complexity to the study, we elected to examine only the middle portion of the heart. Although we believe that similar conclusions would have been reached by examining other regions of the heart, it is realized that this can not be stated with certainty in the absence of supportive data. A number of approaches were examined in pilot studies to address the potential problem of distinguishing individual myocytes from doublets or multicellular clumps in the KOH-digested preps. It was concluded that the most efficient and consistent manner to deal with this problem was to eliminate
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any myocytes containing three or more nuclei. Since these cells comprised less than 2% of the population of rat left-ventricular myocytes, any resulting error in measurements would be negligible. It should also be noted that observers could usually recognize intercalated discs between multiple cells. An additional aid in recognition of multiple cells was the irregular, stair-stepped transition from one cell to the next at intercalated discs. It has been suggested that “myocyte slippage” plays a major role in progressive chamber dilation leading to failure (Linzbach, 1976; Olivetti et al., 1990) though much of the literature mentioning this phenomenon is rather vague. This concept usually refers to slippage of myocytes past one another transversely or linear slippage of individual myofibrils within myocytes (Linzbach, 1976; Komamura et al., 1993). It would seem more likely that linear slippage of myocytes past one another could occur only at fascial planes due to the complex interdigitating connections between myocytes and the presence of intermyocyte collagen struts (Hoyt et al., 1989). Unfortunately, it is difficult to quantitate myocyte slippage directly. Linzbach (1976) and others (Olivetti et al., 1990) have noted a reduction in the number of myocytes across the wall as evidence of myocyte slippage. In our hands, we have found this approach to be subjective and inconsistent. Loss of Z-line registration has also been cited as evidence supporting myocyte slippage in various models of cardiac hypertrophy and failure (Ross et al., 1971; Papadimitriou et al., 1974; Hatt et al., 1979; Komamura et al., 1993; Sharov et al., 1994). While the evidence for this phenomenon appears to be strong, it should be noted that variation in sectioning angle or non-uniform fixation of sarcomeres can produce similar changes. In our experience, we have not observed a significant loss of sarcomere registration in any model of hypertrophy or failure examined to date. This includes the recent examination of six perfusion-fixed hearts obtained from SHHF rats with heart failure (unpublished observation). This study clearly demonstrates that myocyte length and sarcomere length can account for changes in chamber circumference leading to heart failure from hypertension. Although data submitted here do not completely exclude the possibility of myocyte slippage in this model, it appears that any such contributions are relatively insignificant and would be very difficult to detect. It should be realized that fiber direction changes across the wall much like a Japanese fan (Streeter and Hanna, 1973; Pearlman et al., 1982). While
the mid-myocardial portion of the ventricular wall (approximately half of the wall thickness) generally runs in a circumferential direction within transverse slices, measurements were also collected from epicardial and endocardial cells arranged obliquely or perpendicular to the plane of reference. If significant transmural differences in myocyte length were present across the wall, measurements could have been adversely affected. This is a very unlikely possibility, however, since we have not observed significant transmural differences in myocyte length in any of the models of cardiac hypertrophy examined in our lab over the years.
Acknowledgements This work was supported by grant HL 30696 from the National Institutes of Health. We are grateful to Dr Scott Campbell, Carrie Kline and Misty Smith for technical assistance.
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