Number 21, 2003
Hibernation preconditioning

Cellular changes in hypertrophic cardiomyopathy

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Charles S. Redwood
Department of Cardiovascular Medicine, University of Oxford, Wellcome Trust Centre for Human Genetics, Oxford, UK
Correspondence: Dr Charles S. Redwood, Department of Cardiovascular Medicine, University of Oxford, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, UK.
Tel: +441865287663, fax: +441865287586, e-mail: credwood@well.ox.ac.uk

Hypertrophic cardiomyopathy is caused by mutations in sarcomeric protein genes
Hypertrophic cardiomyopathy (HCM) is an autosomal dominant inherited disorder affecting up to 1 in 500 of the population. It is characterized by unexplained asymmetric left ventricular hypertrophy and by myocyte disarray. A groundbreaking paper from the Seidman laboratory, published in 1990, was the first to report the identification of a specific mutant allele that causes HCM [1]. The affected gene was identified as MYH7, which encodes β-myosin heavy chain (βMyHC), the motor protein of the cardiac sarcomere, and the reported missense mutation resulted in an arginine to glutamine amino acid substitution at residue 403. Since this seminal report, at least 70 further HCM mutations in this gene have been reported, accounting for about one third of all affected individuals; in addition, by either linkage analysis or candidate gene screening, at least eight further disease genes that encode components of either the thick myosin filaments (most commonly cardiac myosin binding protein-C [MyBPC]) or the thin actin filaments (principally cardiac troponin T, a component of the trimeric troponin complex, which is responsible for the calcium regulation of contractility) have been identified (see Refresher Corner in this issue). This has led to HCM being termed a disease of the sarcomere [2], in which the primary defect caused by the mutations is an alteration in the normal contraction or relaxation, or both, of cardiac muscle, giving rise to stimuli that promote cellular hypertrophy and ventricular remodeling.
Numerous biophysical, biochemical, and physiological studies have sought to determine the fundamental changes in contractility caused by HCM gene mutations [3]. Initial work focused on the βMyHC mutant proteins, which, in a number of studies using skinned muscle fibers and in-vitro sliding filament assays, were found to be less efficient motors than wild-type myosin, leading to lower sliding velocity and reduced force [4]. These findings led to the proposal that the hypertrophy produced by these mutations was compensatory, in response to the lower force generated by the mutant sarcomeres [5]. More recent work, however, has suggested that these βMyHC mutants are able to translocate actin filaments at faster (not slower) velocities, bringing some of the earlier data into question [4]. Furthermore, functional analysis of mutant alleles of other hypertrophic cardiomyopathy genes has suggested that these are likely to act via a “hypercontractile” mechanism. For example, most HCM mutations in cardiac troponin T and other thin filament proteins appear to cause an increase in the calcium sensitivity of the regulation of contraction, thus causing increased force at any submaximal calcium concentration, and relaxation abnormalities [6].

From contractile abnormality to stimulation of hypertrophy
Studies of models of acquired cardiac hypertrophy (for example via pressure overload) have suggested that a number of interrelated signaling pathways, including the mitogen-activated protein kinases, calmodulin-dependent protein kinase II (CaMKII), and calcineurin, are activated (Figure 1) [7–9]. Is one or more of these pathways triggered by the contractile changes brought about by the HCM mutant proteins?

As the cytoplasmic Ca²⁺ concentration, [Ca²⁺]_i, is intimately linked to the regulation of contractility, it has been hypothesized that the HCM may cause Ca²⁺ dysregulation [10], possibly leading to increased [Ca²⁺]_i and activation of the Ca²⁺/calmodulin-sensitive phosphatase calcineurin, CaMKII, or Ca²⁺-sensitive protein kinase C isoforms (Figure 1). There is some, albeit limited, experimental evidence for this in mouse models of HCM; for example, ventricular myocytes paced at physiological rates from Ile79Asn mutant troponin T hearts showed increased diastolic [Ca²⁺]_i compared with those from nontransgenic animals. In most animal models, increases in diastolic [Ca²⁺]_i have not been reported, although small but significant changes in the resting concentration are difficult to detect using the common spectroscopic indicators (eg, fura-2). Furthermore, the [Ca²⁺]_i surrounding the contractile apparatus varies over a 10-fold range approximately once a second, as a result of the cardiac contraction-relaxation cycle, suggesting that sustained small increases in [Ca²⁺]_i may be sensed in a distinct subcellular pool. The mechanism of CaMKII regulation, however, permits its sustained activation, as it undergoes autophosphorylation, which maintains the enzyme in its active form [11].
It has been suggested that increases in [Ca²⁺]_i may be caused by “calcium trapping” by the mutant sarcomere [12], mediated by changes in the Ca²⁺ affinity of troponin C, widely believed to be the major Ca²⁺ buffer during the Ca²⁺ transient [13]. It is well established that the HCM mutants in troponin T, troponin I and α-tropomyosin in general result in increased myofilament calcium sensitivity, which is likely to be mediated by an increase in the low-affinity, regulatory site of troponin C [14]. In addition, “hypercontractile” βMyHC or MyBPC mutants give an increase in strong myosin head binding, which in turn would increase Ca²⁺ binding by troponin C. A slower release of the “trapped” calcium at the end of the transient would reduce the efficiency of reuptake by the sarcoplasmic reticulum, resulting in both a heightened diastolic [Ca²⁺]_i and a reduced sarcoplasmic reticular Ca²⁺ load. It is important to note, however, that increases in thin-filament Ca²⁺ sensitivity do not necessarily result in hypertrophy; for example, cardiomyocytes from mice expressing the slow skeletal isoform of troponin I show increased Ca²⁺ sensitivity, but the hearts appear to have normal morphology [15].
Another postulated mechanism resulting in calcium dysregulation involves energetic compromise [16]. The myosin ATPase accounts for at least 70% of ATP hydrolysis in the cardiac myocyte, and perturbation of either the motor itself or its regulation may alter the efficiency of ATP usage by the sarcomere; this is supported by data showing increased tension cost in mutant mouse fibers [17]. Chronic increase in the energy cost of maintaining normal power output will give rise to ATP depletion, which is predicted to affect other highly ATP-dependent processes in the cell, such as the sarcoplasmic reticulum calcium pump (SERCA), the ATPase with the greatest minimal energy requirement in muscle cells [18]. There is strong evidence that energy compromise occurs in the hearts of patients with HCM; a recent paper described similar (approximately 30%) reductions in phosphocreatine:ATP ratios in patients with mutations in βMyHC, MyBPC, or troponin T, in some cases before the development of hypertrophy [19]. In addition, in a mouse model of the original Arg403Gln myosin mutation, a comparable reduction in phosphocreatine:ATP ratio was measured, and it was suggested that the free energy change for ATP hydrolysis was decreased to close to the minimal requirement for SERCA [20]. Further weight has been added to this hypothesis by the finding that HCM in combination with Wolff-Parkinson-White syndrome is caused by mutations in PRKAG2, which encodes the γ2 subunit of AMP-activated protein kinase (AMPK) [21]. This kinase, an αβγ trimer of which α is the catalytic subunit, is activated on ATP depletion, turning on energy-producing pathways (such as glycolysis) and inhibiting ATP-consuming processes (such as fatty acid synthesis), and has been termed the fuel gauge of the cell [22]. Experimental evidence concerning the functional effects of the PRKAG2 mutations is equivocal: analysis of the equivalent amino acid substitutions in the γ1 subunit [23] and in the yeast homologue, Snf1/Snf4 kinase [24], has suggested they may cause either increased kinase activity or constitutive activation, but cellular coexpression of α and β AMPK subunits with either wild-type or mutant γ2 has shown that the mutations result in reduced maximal activity or lower AMP dependence, or both [25]. Clearly, a reduction in the ability to sense low concentrations of ATP may lead to compromised SERCA activity, by the mechanism described above. Recent studies have also suggested that AMPK may directly interact with components of hypertrophic signaling pathways such as Akt and other downstream targets, and suppress translation [26–28], implying that reduction in AMPK activity may increase translation and act to promote hypertrophy.
Attempts to inhibit calcineurin using cyclosporin A (CsA) in the Arg403Gln myosin mouse model gave the somewhat confusing result of enhanced hypertrophy [12]. In addition, the increase in diastolic [Ca²⁺]_i caused by CsA in wild-type animals was absent, clearly demonstrating Ca²⁺ dysregulation in the mutant mouse hearts. It is established that there is considerable crosstalk between calcineurin and other hypertrophic signaling pathways [29], and there are other instances of inhibition of a known hypertrophic mediator producing an unexpected result (eg, inhibition of p38 MAPK promoting hypertrophy [30]). In the sarcoplasmic reticulum of the Arg403Gln myosin mutant mouse heart, there is reduced Ca²⁺ load and concentrations of both calsequestrin and ryanidine receptor are diminished, leading to the suggestion that disruption of sarcoplasmic reticular Ca²⁺ homeostasis is a key mediator of the pathogenic process [31]. Interestingly, in this model the administration of the L-type Ca²⁺ channel inhibitor, diltiazem, restored to normal the concentrations of the sarcoplasmic reticulum proteins, and prevented the development of hypertrophy.

Summary
Many of the effects on contractility produced by the sarcomeric protein gene mutations are understood, but the exact nature of the signaling pathways responsible for the generation of pathophysiological hypertrophy in HCM remains to be elucidated. It seems likely, however, that both disruption of Ca²⁺ homeostasis and energetic compromise are involved as triggers. It is hoped that future work will identify the exact changes involved, and that such findings may suggest the possibility of appropriately targeted pharmacological intervention. ▪

Acknowledgments
This work was supported by the British Heart Foundation. The author thanks Hend Farza, Edward Blair, and Hugh Watkins for helpful discussions on the manuscript.

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REFERENCES

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